renaming BioC XML files to show PMC ID as file name and to be able to distinguish between raw and annotated XML; add folder with JSON version of annotated BioC

LICENSE.md

@@ -0,0 +1,9 @@
+MIT License
+
+Copyright (c) 2023, Melanie Vollmar
+
+Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the “Software”), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:
+
+The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.
+
+THE SOFTWARE IS PROVIDED “AS IS”, WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.

annotated_BioC_JSON/PMC4854314_ann.json

@@ -0,0 +1 @@
+[{"sourceid":"4854314","sourcedb":"","project":"","target":"","text":"RNA protects a nucleoprotein complex against radiation damage Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. A new method of difference electron-density quantification is presented. Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. Introduction    With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov \u0026 Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved. RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray \u0026 Garman, 2002). Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kg−1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006).  Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli \u0026 McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005). Furthermore, damage susceptibility within each residue type follows a preferential ordering influenced by a combination of local environment factors (solvent accessibility, conformational strain, proximity to active sites/high X-ray cross-section atoms; Holton, 2009). Deconvoluting the individual roles of these parameters has been surprisingly challenging, with factors such as solvent accessibility currently under active investigation (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism. For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005). Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. As of early 2016, \u003e5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them. Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments. When a typical macromolecular crystal is irradiated with ionizing X-rays, each photoelectron produced via interactions with both the macromolecule (direct damage) and solvent (indirect damage) can induce cascades of up to 500 secondary low-energy electrons (LEEs) that are capable of inducing further ionizations. Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh \u0026 Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska \u0026 Sanche, 2007). Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002). The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA. For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP. Materials and methods    RNA synthesis and protein preparation    As previously described (Hopcroft et al., 2002), the 53-base RNA (GAGUU)10GAG was synthesized by in vitro transcription with T7 RNA polymerase and gel-purified. TRAP from B. stearothermophilus was overexpressed in Escherichia coli and purified. Crystallization    TRAP–RNA crystals were prepared using a previously established hanging-drop crystallization protocol (Antson et al., 1999). By using a 2:1 molar ratio of TRAP to RNA, crystals successfully formed from the protein–RNA complex (∼15 mg ml−1) in a solution containing 70 mM potassium phosphate pH 7.8 and 10 mM l-tryptophan. The reservoir consisted of 0.2 M potassium glutamate, 50 mM triethanol­amine pH 8.0, 10 mM MgCl2, 8–11% monomethyl ether PEG 2000. In order to accelerate crystallization, a further gradient was induced by adding 0.4 M KCl to the reservoir after 1.5 µl protein solution had been mixed with an equal volume of the reservoir solution. Wedge-shaped crystals of approximate length 70 µm (longest dimension) grew within 3 d and were vitrified and stored in liquid nitrogen immediately after growth. The cryosolution consisted of 12% monomethyl ether PEG 2000, 30 mM triethanolamine pH 8.0, 6 mM l-tryptophan, 0.1 M potassium glutamate, 35 mM potassium phosphate pH 7.8, 5 mM MgCl2 with 25% 2-methyl-2,4-pentanediol (MPD) included as a cryoprotectant. X-ray data collection    Data were collected at 100 K from a wedge-shaped TRAP–RNA crystal of approximate dimensions 70 × 20 × 40 µm (see Supplementary Fig. S2) on beamline ID14-4 at the ESRF using an incident wavelength of 0.940 Å (13.2 keV) and an ADSC Q315R mosaic CCD detector at 304.5 mm from the crystal throughout the data collection. The beam size was slitted to 0.100 mm (vertical) × 0.160 mm (horizontal), with a uniformly distributed profile, such that the crystal was completely bathed within the beam throughout data collection. Ten successive (1.98 Å resolution) 180° data sets (with Δφ = 1°) were collected over the same angular range from a TRAP–RNA crystal at 28.9% beam transmission. The TRAP–RNA macromolecule crystallized in space group C2, with unit-cell parameters a = 140.9, b = 110.9, c = 137.8 Å, α = γ = 90, β = 137.8° (the values quoted are for the first data set; see Supplementary Table S1 for subsequent values). For the first nine data sets the attenuated flux was recorded to be ∼5 × 1011 photons s−1. A beam refill took place immediately before data set 10, requiring a flux-scale factor increase of 1.42 to be applied, based on the ratio of observed relative intensity I D/I 1 at data set 10 to that extrapolated from data set 9. Dose calculation     RADDOSE-3D (Zeldin, Gerstel et al., 2013) was used to calculate the absorbed dose distribution during each data set (see input file; Supplementary Figs. S1 and S2). The crystal composition was calculated from the deposited TRAP–RNA structure (PDB entry 1gtf; Hopcroft et al., 2002). Crystal absorption coefficients were calculated in RADDOSE-3D using the concentration (mmol l−1) of solvent heavy elements from the crystallization conditions. The beam-intensity profile was modelled as a uniform (‘top-hat’) distribution. The diffraction-weighted dose (DWD) values (Zeldin, Brock­hauser et al., 2013) are given in Supplementary Table S1. Data processing and model refinement    Each data set was integrated using iMosflm (Leslie \u0026 Powell, 2007) and was scaled using AIMLESS (Evans \u0026 Murshudov, 2013; Winn et al., 2011) using the same 5% R free set of test reflections for each data set. To phase the structure obtained from the first data set, molecular replacement was carried out with Phaser (McCoy et al., 2007), using an identical TRAP–RNA structure (PDB entry 1gtf; resolution 1.75 Å; Hopcroft et al., 2002) as a search model. The resulting TRAP–RNA structure (TR1) was refined using REFMAC5 (Murshudov et al., 2011), initially using rigid-body refinement, followed by repeated cycles of restrained, TLS and isotropic B-factor refinement, coupled with visual inspection in Coot (Emsley et al., 2010). TR1 was refined to 1.98 Å resolution, with a dimeric assembly of non-RNA-bound and RNA-bound TRAP rings within the asymmetric unit. Consistent with previous structures of the TRAP–RNA complex, the RNA sequence termini were not observed within the 2F o − F c map; the first spacer (U4) was then modelled at all 11 repeats around the TRAP ring and the second spacer (U5) was omitted from the final refined structure. For the later data sets, the observed structure-factor amplitudes from each separately scaled data set (output from AIMLESS) were combined with the phases of TR1 and the resulting higher-dose model was refined with phenix.refine (Adams et al., 2010) using only rigid-body and isotropic B-factor refinement. During this refinement, the TRAP–RNA complex and nonbound TRAP ring were treated as two separate rigid bodies within the asymmetric unit. Supplementary Table S1 shows the relevant summary statistics.  D loss metric calculation    The CCP4 program CAD was used to create a series of nine merged .mtz files combining observed structure-factor amplitudes for the first data set F obs(d 1) with each later data set F obs(d n) (for n = 2, …, 10). All later data sets were scaled against the initial low-dose data set in SCALEIT. For each data set an atom-tagged .map file was generated using the ATMMAP mode in SFALL (Winn et al., 2011). A full set of nine Fourier difference maps F obs(d n) − F obs(d 1) were calculated using FFT (Ten Eyck, 1973) over the full TRAP–RNA unit-cell dimensions, with the same grid-sampling dimensions as the atom-tagged .map file. All maps were cropped to the TRAP asymmetric unit in MAPMASK. Comparing the atom-tagged .map file and F obs(d n) − F obs(d 1) difference map at each dose, each refined atom was assigned a set of density-change values X. The maximum density-loss metric, D loss (units of e Å−3), was calculated to quantify the per-atom electron-density decay at each dose, assigned as the absolute magnitude of the most negative Fourier difference map voxel value in a local volume around each atom as defined by the set X. Model system calculation    Model calculations were run for the simple amino acids glutamate and aspartate. In order to avoid decarboxylation at the C-terminus instead of the side chain on the Cα atom, the C-terminus of each amino acid was methylated. While the structures of the closed shell acids are well known, the same is not true of those in the oxidized state. The quantum-chemical calculations employed were chosen to provide a satisfactory description of the structure of such radical species and also provide a reliable estimation of the relative C—C(O2) bond strengths, which are otherwise not available. Structures of methyl-terminated (at the N- and C-termini) carboxylates were determined using analytic energy gradients with density functional theory (B3LYP functional; Becke, 1993) and a flexible basis set of polarized valence triple-zeta size with diffuse functions on the non-H atoms [6-311+G(d,p)] in the Gaussian 09 computational chemistry package (Frisch et al., 2009). The stationary points obtained were characterized as at least local minima by examination of the associated analytic Hessian. Effects of the medium were modelled using a dielectric cavity approach (Tomasi et al., 1999) parameterized for water. Results    Per-atom quantification of electron density    To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. This provides an atom-specific quantification of density–dose dynamics, which was previously lacking within the field. Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set. For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. Additionally, the density surrounding ordered solvent molecules was determined to significantly diminish with increasing dose (Fig. 2 ▸ b). The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex    Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). Only at the highest doses investigated (\u003e20 MGy) was density loss observed at the RNA phosphate and C—O bonds of the phosphodiester backbone. However, the median D loss was lower by a factor of \u003e2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RNA binding protects radiation-sensitive residues    For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RNA binding reduces radiation-induced disorder on the atomic scale    One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues. The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). Representative Phe32 and Lys37 atoms were selected to illustrate these trends. Discussion    Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX. Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (\u003e20 MGy). The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred. RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of \u003c0.23 for all doses; Supplementary Fig. S5). Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. Radiation-induced decarboxylation has been proposed to be mediated by preferential positive-hole migration to the side-chain carboxyl group, with rapid proton transfer trapping the hole at the carboxyl group (Burmeister, 2000; Symons, 1997):where the forward rate is K 1 and the backward rate is K −1, where the forward rate is K 2. When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. Previous studies have reported inconsistent results concerning the dependence of the acidic residue decarboxylation rate on solvent accessibility (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al., 2013; Owen et al., 2012). Furthermore, the suggested electron hole-trapping mechanism which induces decarboxylation within proteins at 100 K has no clear mechanistic dependence on the solvent-accessible area of each carboxyl group. By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. Overall, no direct correlation between solvent accessibility and decarboxylation susceptibility was observed, but it is very clear that inaccessible residues are protected. Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. Thus, the dependence of decarboxylation rates on these interactions could not be established. Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain. Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX. Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic –OH group owing to radiation-induced effects; however, no energetically favourable pathway for –OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies. In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups. The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p \u003e 0.9). An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot \u0026 Davídková, 2011; Chance et al., 1997). It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT. However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways. The Python scripts written to calculate the per atom D loss metric are available from the authors on request. Related literature    The following references are cited in the Supporting Information for this article: Chen et al. (2010). Supplementary Material References Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221. Alizadeh, E. \u0026 Sanche, L. (2014). Eur. Phys. J. D, 68, 97. Allan, E. G., Kander, M. C., Carmichael, I. \u0026 Garman, E. F. (2013). J. Synchrotron Rad. 20, 23–36. Antson, A. A., Dodson, E. J., Dodson, G., Greaves, R. B., Chen, X. \u0026 Gollnick, P. (1999). Nature (London), 401, 235–242. Becke, A. D. (1993). J. Chem. Phys. 98, 5648–5652. Bourenkov, G. P. \u0026 Popov, A. N. (2010). Acta Cryst. D66, 409–419. Burmeister, W. P. (2000). Acta Cryst. D56, 328–341. Bury, C., Garman, E. F., Ginn, H. M., Ravelli, R. B. G., Carmichael, I., Kneale, G. \u0026 McGeehan, J. E. (2015). J. Synchrotron Rad. 22, 213–224. Chance, M. R., Sclavi, B., Woodson, S. A. \u0026 Brenowitz, M. (1997). Structure, 5, 865–869. Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. \u0026 Richardson, D. C. (2010). Acta Cryst. D66, 12–21. Dubnovitsky, A. P., Ravelli, R. B. G., Popov, A. N. \u0026 Papageorgiou, A. C. (2005). Protein Sci. 14, 1498–1507. Elliott, M. B., Gottlieb, P. A. \u0026 Gollnick, P. (2001). RNA, 7, 85–93. Emsley, P., Lohkamp, B., Scott, W. G. \u0026 Cowtan, K. (2010). Acta Cryst. D66, 486–501. Evans, P. R. \u0026 Murshudov, G. N. (2013). Acta Cryst. D69, 1204–1214. Fioravanti, E., Vellieux, F. M. D., Amara, P., Madern, D. \u0026 Weik, M. (2007). J. Synchrotron Rad. 14, 84–91. Frisch, M. J. et al. (2009). Gaussian 09. Gaussian Inc., Wallingford, Connecticut, USA. Garman, E. F. (2010). Acta Cryst. D66, 339–351. Gerstel, M., Deane, C. M. \u0026 Garman, E. F. (2015). J. Synchrotron Rad. 22, 201–212. Gillard, N., Begusova, M., Castaing, B. \u0026 Spotheim-Maurizot, M. (2004). Radiat. Res. 162, 566–571. Holton, J. M. (2007). J. Synchrotron Rad. 14, 51–72. Holton, J. M. (2009). J. Synchrotron Rad. 16, 133–142. Hopcroft, N. H., Wendt, A. L., Gollnick, P. \u0026 Antson, A. A. (2002). Acta Cryst. D58, 615–621. Jones, G. D., Lea, J. S., Symons, M. C. \u0026 Taiwo, F. A. (1987). Nature (London), 330, 772–773. Leslie, A. G. W. \u0026 Powell, H. R. (2007). Evolving Methods for Macromolecular Crystallography, edited by R. J. Read \u0026 J. L. Sussman, pp. 41–51. Dordrecht: Springer. Liebschner, D., Rosenbaum, G., Dauter, M. \u0026 Dauter, Z. (2015). Acta Cryst. D71, 772–778. Matsui, Y., Sakai, K., Murakami, M., Shiro, Y., Adachi, S., Okumura, H. \u0026 Kouyama, T. (2002). J. Mol. Biol. 324, 469–481. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. \u0026 Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. McGeehan, J. E., Streeter, S. D., Thresh, S. J., Ball, N., Ravelli, R. B. G. \u0026 Kneale, G. G. (2008). Nucleic Acids Res. 36, 4778–4787. Murray, J. \u0026 Garman, E. (2002). J. Synchrotron Rad. 9, 347–354. Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. \u0026 Vagin, A. A. (2011). Acta Cryst. D67, 355–367. O’Neill, P., Stevens, D. L. \u0026 Garman, E. (2002). J. Synchrotron Rad. 9, 329–332. Owen, R. L., Axford, D., Nettleship, J. E., Owens, R. J., Robinson, J. I., Morgan, A. W., Doré, A. S., Lebon, G., Tate, C. G., Fry, E. E., Ren, J., Stuart, D. I. \u0026 Evans, G. (2012). Acta Cryst. D68, 810–818. Owen, R. L., Rudiño-Piñera, E. \u0026 Garman, E. F. (2006). Proc. Natl Acad. Sci. USA, 103, 4912–4917. Ptasińska, S. \u0026 Sanche, L. (2007). Phys. Rev. E, 75, 031915. Ravelli, R. B. G. \u0026 McSweeney, S. M. (2000). Structure, 8, 315–328. Shimizu, N., Hirata, K., Hasegawa, K., Ueno, G. \u0026 Yamamoto, M. (2007). J. Synchrotron Rad. 14, 4–10. Simons, J. (2006). Acc. Chem. Res. 39, 772–779. Spotheim-Maurizot, M. \u0026 Davídková, M. (2011). Mutat. Res. 711, 41–48. Stísová, V., Goffinont, S., Spotheim-Maurizot, M. \u0026 Davídková, M. (2006). Radiat. Prot. Dosimetry, 122, 106–109. Symons, M. C. R. (1997). Free Radical Biol. Med. 22, 1271–1276. Ten Eyck, L. F. (1973). Acta Cryst. A29, 183–191. Tomasi, J., Mennucci, B. \u0026 Cancès, E. (1999). J. Mol. Struct. 464, 211–226. Weik, M., Ravelli, R. B. G., Kryger, G., McSweeney, S., Raves, M. L., Harel, M., Gros, P., Silman, I., Kroon, J. \u0026 Sussman, J. L. (2000). Proc. Natl Acad. Sci. USA, 97, 623–628. Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. Yano, J., Kern, J., Irrgang, K. D., Latimer, M. J., Bergmann, U., Glatzel, P., Pushkar, Y., Biesiadka, J., Loll, B., Sauer, K., Messinger, J., Zouni, A. \u0026 Yachandra, V. K. (2005). Proc. Natl Acad. Sci. USA, 102, 12047–12052. Zeldin, O. B., Brockhauser, S., Bremridge, J., Holton, J. M. \u0026 Garman, E. F. (2013). Proc. Natl Acad. Sci. USA, 110, 20551–20556. Zeldin, O. B., Gerstel, M. \u0026 Garman, E. F. (2013). J. Appl. Cryst. 46, 1225–1230. The TRAP–(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002). Bound tryptophan ligands are represented as coloured spheres. RNA is shown is yellow. (a) Electron-density loss sites as indicated by D\nloss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D\nloss threshold, calculated over the TRAP–RNA structure for the first difference map: F\nobs(d\n2) − F\nobs(d\n1)]. Cumulative frequencies are normalized to both the total number of non-H atoms per residue/nucleotide and the total number of that residue/nucleotide type present. (b) Average D\nloss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). 95% confidence intervals (CI) are shown. Only a subset of key TRAP residue types are included. The average D\nloss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line). \nF\nobs(d\nn) − F\nobs(d\n1) Fourier difference maps for (a) n = 2 (3.9 MGy), (b) n = 3 (6.5 MGy) and (c) n = 7 (16.7 MGy) contoured at ±4σ (a) and ±3.5σ (b, c). In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d\n2 = 3.9 MGy. Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed. \nD\nloss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d\n8). Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. Whiskers indicate 95% CI. The D\nloss behaviour shown here was consistently exhibited across the entire investigated dose range. \nD\nloss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. RNA-binding interface interactions are shown for TRAP chain N, with the F\nobs(d\n7) − F\nobs(d\n1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ level.","denotations":[{"span":{"begin":0,"end":3},"obj":"0.9980228,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"1"},{"span":{"begin":15,"end":28},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T10:34:01Z","id":"760"},{"span":{"begin":111,"end":122},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:04:56Z","id":"839"},{"span":{"begin":219,"end":222},"obj":"0.9902272,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"3"},{"span":{"begin":252,"end":294},"obj":"0.9882428,experimental_method,cleaner0,2023-07-05T12:16:13Z,MESH:","id":"4"},{"span":{"begin":339,"end":392},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:16:36Z","id":"887"},{"span":{"begin":431,"end":470},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T10:26:50Z","id":"725"},{"span":{"begin":803,"end":811},"obj":"0.93006164,evidence,cleaner0,2023-07-05T10:27:59Z,DUMMY:","id":"7"},{"span":{"begin":960,"end":984},"obj":"0.9697803,evidence,cleaner0,2023-07-05T10:28:01Z,DUMMY:","id":"8"},{"span":{"begin":1157,"end":1160},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"763"},{"span":{"begin":1234,"end":1269},"obj":"0.9563537,protein_type,cleaner0,2023-07-05T12:26:59Z,MESH:","id":"11"},{"span":{"begin":1271,"end":1275},"obj":"0.53643787,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"12"},{"span":{"begin":1277,"end":1285},"obj":"0.99148065,protein_state,cleaner0,2023-07-05T12:33:37Z,DUMMY:","id":"13"},{"span":{"begin":1304,"end":1307},"obj":"0.9971852,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"15"},{"span":{"begin":1375,"end":1378},"obj":"0.99032986,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"16"},{"span":{"begin":1496,"end":1500},"obj":"0.937143,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"17"},{"span":{"begin":1563,"end":1568},"obj":"0.9731102,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"18"},{"span":{"begin":1569,"end":1572},"obj":"0.99720335,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"19"},{"span":{"begin":1632,"end":1635},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"669"},{"span":{"begin":1720,"end":1724},"obj":"0.8899272,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"20"},{"span":{"begin":1725,"end":1729},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"821"},{"span":{"begin":1782,"end":1785},"obj":"0.9860465,residue_name,cleaner0,2023-07-05T10:28:06Z,SO:","id":"21"},{"span":{"begin":1790,"end":1793},"obj":"0.9869811,residue_name,cleaner0,2023-07-05T10:28:09Z,SO:","id":"22"},{"span":{"begin":1816,"end":1819},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"670"},{"span":{"begin":1923,"end":1942},"obj":"0.9977442,site,cleaner0,2023-07-05T12:01:06Z,SO:","id":"23"},{"span":{"begin":2087,"end":2090},"obj":"0.9823989,residue_name,cleaner0,2023-07-05T10:28:11Z,SO:","id":"24"},{"span":{"begin":2095,"end":2098},"obj":"0.9763282,residue_name,cleaner0,2023-07-05T10:28:13Z,SO:","id":"25"},{"span":{"begin":2116,"end":2119},"obj":"0.652342,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"26"},{"span":{"begin":2146,"end":2162},"obj":"0.9961912,evidence,cleaner0,2023-07-05T10:28:04Z,DUMMY:","id":"27"},{"span":{"begin":2340,"end":2363},"obj":"0.8348757,experimental_method,cleaner0,2023-07-05T10:28:23Z,MESH:","id":"28"},{"span":{"begin":2370,"end":2400},"obj":"0.98041725,experimental_method,cleaner0,2023-07-05T10:28:25Z,MESH:","id":"29"},{"span":{"begin":2402,"end":2404},"obj":"0.56438595,experimental_method,cleaner0,2023-07-05T10:28:29Z,MESH:","id":"30"},{"span":{"begin":2630,"end":2638},"obj":"0.9865131,evidence,cleaner0,2023-07-05T10:28:34Z,DUMMY:","id":"31"},{"span":{"begin":2888,"end":2890},"obj":"0.9509965,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"32"},{"span":{"begin":3283,"end":3302},"obj":"0.9363032,evidence,cleaner0,2023-07-05T12:19:18Z,DUMMY:","id":"33"},{"span":{"begin":3534,"end":3536},"obj":"0.96844155,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"34"},{"span":{"begin":3732,"end":3739},"obj":"0.91692525,evidence,cleaner0,2023-07-05T10:31:01Z,DUMMY:","id":"35"},{"span":{"begin":3793,"end":3818},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:19:00Z","id":"892"},{"span":{"begin":3820,"end":3823},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:19:10Z","id":"893"},{"span":{"begin":3844,"end":3871},"obj":"0.9674265,evidence,cleaner0,2023-07-05T10:31:05Z,DUMMY:","id":"36"},{"span":{"begin":3988,"end":3990},"obj":"0.28211054,chemical,cleaner0,2023-07-05T10:29:47Z,CHEBI:","id":"37"},{"span":{"begin":3999,"end":4015},"obj":"0.9843067,chemical,cleaner0,2023-07-05T10:29:51Z,CHEBI:","id":"38"},{"span":{"begin":4143,"end":4150},"obj":"0.9706023,evidence,cleaner0,2023-07-05T10:31:07Z,DUMMY:","id":"39"},{"span":{"begin":4319,"end":4333},"obj":"ptm,MESH:,cleaner0,2023-07-05T10:30:47Z","id":"757"},{"span":{"begin":5120,"end":5133},"obj":"0.9724948,experimental_method,cleaner0,2023-07-05T10:31:22Z,MESH:","id":"45"},{"span":{"begin":5134,"end":5144},"obj":"0.9942268,evidence,cleaner0,2023-07-05T12:19:53Z,DUMMY:","id":"46"},{"span":{"begin":5267,"end":5287},"obj":"0.9978055,site,cleaner0,2023-07-05T12:01:17Z,SO:","id":"47"},{"span":{"begin":5472,"end":5495},"obj":"0.99299383,experimental_method,cleaner0,2023-07-05T10:31:25Z,MESH:","id":"48"},{"span":{"begin":5527,"end":5545},"obj":"0.84919345,protein_type,cleaner0,2023-07-05T10:31:44Z,MESH:","id":"49"},{"span":{"begin":5777,"end":5788},"obj":"0.99806416,site,cleaner0,2023-07-05T12:01:21Z,SO:","id":"50"},{"span":{"begin":5792,"end":5822},"obj":"0.9870368,protein_type,cleaner0,2023-07-05T10:31:34Z,MESH:","id":"51"},{"span":{"begin":5928,"end":5939},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T10:32:31Z","id":"758"},{"span":{"begin":6066,"end":6079},"obj":"0.5266972,complex_assembly,cleaner0,2023-07-05T11:49:23Z,GO:","id":"56"},{"span":{"begin":6208,"end":6211},"obj":"0.6261449,chemical,cleaner0,2023-07-05T10:36:34Z,CHEBI:","id":"57"},{"span":{"begin":6348,"end":6351},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"764"},{"span":{"begin":6384,"end":6397},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T10:33:26Z","id":"759"},{"span":{"begin":6406,"end":6416},"obj":"0.9936459,evidence,cleaner0,2023-07-05T12:19:56Z,DUMMY:","id":"59"},{"span":{"begin":6472,"end":6474},"obj":"0.9480319,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"60"},{"span":{"begin":6552,"end":6562},"obj":"0.9201258,evidence,cleaner0,2023-07-05T12:19:59Z,DUMMY:","id":"61"},{"span":{"begin":6613,"end":6627},"obj":"0.78504515,complex_assembly,cleaner0,2023-07-05T10:33:41Z,GO:","id":"62"},{"span":{"begin":7303,"end":7306},"obj":"0.9642742,chemical,cleaner0,2023-07-05T10:36:34Z,CHEBI:","id":"65"},{"span":{"begin":7464,"end":7467},"obj":"0.94849676,chemical,cleaner0,2023-07-05T10:36:34Z,CHEBI:","id":"66"},{"span":{"begin":7639,"end":7675},"obj":"0.99424225,experimental_method,cleaner0,2023-07-05T10:34:36Z,MESH:","id":"67"},{"span":{"begin":7834,"end":7837},"obj":"0.8569334,chemical,cleaner0,2023-07-05T10:36:34Z,CHEBI:","id":"68"},{"span":{"begin":7954,"end":7970},"obj":"0.97740066,site,cleaner0,2023-07-05T12:01:27Z,SO:","id":"69"},{"span":{"begin":8216,"end":8228},"obj":"0.92757994,experimental_method,cleaner0,2023-07-05T10:35:56Z,MESH:","id":"70"},{"span":{"begin":8276,"end":8285},"obj":"taxonomy_domain,DUMMY:,cleaner0,2023-07-05T10:35:49Z","id":"761"},{"span":{"begin":8294,"end":8297},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"765"},{"span":{"begin":8307,"end":8317},"obj":"0.99454117,complex_assembly,cleaner0,2023-07-05T12:31:03Z,GO:","id":"73"},{"span":{"begin":8489,"end":8492},"obj":"0.99493873,chemical,cleaner0,2023-07-05T10:36:33Z,CHEBI:","id":"74"},{"span":{"begin":8504,"end":8511},"obj":"0.9964142,evidence,cleaner0,2023-07-05T12:20:04Z,DUMMY:","id":"75"},{"span":{"begin":8624,"end":8639},"obj":"structure_element,SO:,cleaner0,2023-07-05T10:36:28Z","id":"762"},{"span":{"begin":8654,"end":8657},"obj":"0.9971282,chemical,cleaner0,2023-07-05T10:36:34Z,CHEBI:","id":"78"},{"span":{"begin":8704,"end":8714},"obj":"0.99455875,complex_assembly,cleaner0,2023-07-05T12:31:03Z,GO:","id":"80"},{"span":{"begin":8852,"end":8855},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"766"},{"span":{"begin":9094,"end":9101},"obj":"0.9853733,evidence,cleaner0,2023-07-05T12:20:08Z,DUMMY:","id":"82"},{"span":{"begin":9178,"end":9186},"obj":"0.98737174,protein_state,cleaner0,2023-07-05T12:33:43Z,DUMMY:","id":"83"},{"span":{"begin":9187,"end":9190},"obj":"0.9977902,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"84"},{"span":{"begin":9269,"end":9283},"obj":"0.9718507,experimental_method,cleaner0,2023-07-05T12:16:48Z,MESH:","id":"85"},{"span":{"begin":9355,"end":9366},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:04:56Z","id":"840"},{"span":{"begin":9376,"end":9411},"obj":"0.9377823,protein_type,cleaner0,2023-07-05T12:27:03Z,MESH:","id":"88"},{"span":{"begin":9413,"end":9417},"obj":"0.8530336,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"89"},{"span":{"begin":9419,"end":9427},"obj":"0.9948477,protein_state,cleaner0,2023-07-05T11:51:21Z,DUMMY:","id":"90"},{"span":{"begin":9436,"end":9439},"obj":"0.99753857,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"91"},{"span":{"begin":9449,"end":9461},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:38:29Z","id":"776"},{"span":{"begin":9503,"end":9507},"obj":"0.9524646,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"93"},{"span":{"begin":9533,"end":9541},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:54Z","id":"882"},{"span":{"begin":9559,"end":9563},"obj":"0.5846764,structure_element,cleaner0,2023-07-05T11:58:12Z,SO:","id":"94"},{"span":{"begin":9627,"end":9630},"obj":"0.9860402,evidence,cleaner0,2023-07-05T12:20:10Z,DUMMY:","id":"95"},{"span":{"begin":9644,"end":9647},"obj":"0.9952761,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"96"},{"span":{"begin":9671,"end":9687},"obj":"0.9207858,structure_element,cleaner0,2023-07-05T10:47:16Z,SO:","id":"97"},{"span":{"begin":9714,"end":9732},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:54:57Z","id":"820"},{"span":{"begin":9789,"end":9799},"obj":"0.90775585,chemical,cleaner0,2023-07-05T12:14:09Z,CHEBI:","id":"100"},{"span":{"begin":9822,"end":9839},"obj":"0.99550605,species,cleaner0,2023-07-05T10:37:26Z,MESH:","id":"101"},{"span":{"begin":9871,"end":9880},"obj":"0.9971539,evidence,cleaner0,2023-07-05T12:20:16Z,DUMMY:","id":"102"},{"span":{"begin":9899,"end":9907},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:39:38Z","id":"778"},{"span":{"begin":9974,"end":9979},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:39:55Z","id":"779"},{"span":{"begin":10077,"end":10079},"obj":"0.947729,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"107"},{"span":{"begin":10119,"end":10127},"obj":"0.98999834,complex_assembly,cleaner0,2023-07-05T10:38:43Z,GO:","id":"108"},{"span":{"begin":10128,"end":10135},"obj":"0.9876061,evidence,cleaner0,2023-07-05T10:40:23Z,DUMMY:","id":"109"},{"span":{"begin":10297,"end":10318},"obj":"0.99650353,evidence,cleaner0,2023-07-05T10:40:25Z,DUMMY:","id":"114"},{"span":{"begin":10329,"end":10338},"obj":"0.99333715,site,cleaner0,2023-07-05T12:01:33Z,SO:","id":"115"},{"span":{"begin":10394,"end":10417},"obj":"0.9945757,evidence,cleaner0,2023-07-05T10:40:28Z,DUMMY:","id":"116"},{"span":{"begin":10615,"end":10619},"obj":"0.9496573,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"117"},{"span":{"begin":10718,"end":10721},"obj":"0.99639255,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"118"},{"span":{"begin":10733,"end":10737},"obj":"0.9609993,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"119"},{"span":{"begin":18315,"end":18358},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T11:50:30Z","id":"819"},{"span":{"begin":18538,"end":18561},"obj":"0.993571,evidence,cleaner0,2023-07-05T12:20:24Z,DUMMY:","id":"279"},{"span":{"begin":18596,"end":18604},"obj":"0.9888622,complex_assembly,cleaner0,2023-07-05T10:38:43Z,GO:","id":"280"},{"span":{"begin":18605,"end":18614},"obj":"0.9903577,evidence,cleaner0,2023-07-05T12:20:28Z,DUMMY:","id":"281"},{"span":{"begin":18687,"end":18708},"obj":"0.82108414,evidence,cleaner0,2023-07-05T12:20:32Z,DUMMY:","id":"282"},{"span":{"begin":18793,"end":18802},"obj":"0.9954945,site,cleaner0,2023-07-05T12:01:39Z,SO:","id":"283"},{"span":{"begin":18830,"end":18882},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:01:56Z","id":"834"},{"span":{"begin":18899,"end":18904},"obj":"0.88435054,evidence,cleaner0,2023-07-05T12:20:37Z,DUMMY:","id":"286"},{"span":{"begin":18906,"end":18907},"obj":"0.58001226,evidence,cleaner0,2023-07-05T12:20:40Z,DUMMY:","id":"287"},{"span":{"begin":18938,"end":18957},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:03:31Z","id":"837"},{"span":{"begin":18967,"end":18998},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:03:02Z","id":"835"},{"span":{"begin":19046,"end":19047},"obj":"0.7078795,evidence,cleaner0,2023-07-05T12:20:44Z,DUMMY:","id":"289"},{"span":{"begin":19069,"end":19087},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:03:19Z","id":"836"},{"span":{"begin":19102,"end":19105},"obj":"0.9917287,evidence,cleaner0,2023-07-05T12:20:48Z,DUMMY:","id":"291"},{"span":{"begin":19199,"end":19206},"obj":"0.6958162,evidence,cleaner0,2023-07-05T12:20:51Z,DUMMY:","id":"292"},{"span":{"begin":19264,"end":19291},"obj":"0.9909112,evidence,cleaner0,2023-07-05T12:20:55Z,DUMMY:","id":"293"},{"span":{"begin":19293,"end":19299},"obj":"0.97732365,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"294"},{"span":{"begin":19361,"end":19398},"obj":"0.9871848,evidence,cleaner0,2023-07-05T12:21:00Z,DUMMY:","id":"295"},{"span":{"begin":19433,"end":19439},"obj":"0.98676395,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"296"},{"span":{"begin":19601,"end":19609},"obj":"0.96854377,complex_assembly,cleaner0,2023-07-05T10:38:43Z,GO:","id":"298"},{"span":{"begin":19624,"end":19637},"obj":"0.9839508,evidence,cleaner0,2023-07-05T12:21:04Z,DUMMY:","id":"299"},{"span":{"begin":19694,"end":19697},"obj":"0.98396283,experimental_method,cleaner0,2023-07-05T12:27:21Z,MESH:","id":"300"},{"span":{"begin":19723,"end":19726},"obj":"0.9861578,residue_name,cleaner0,2023-07-05T12:07:07Z,SO:","id":"301"},{"span":{"begin":19731,"end":19734},"obj":"0.98857224,residue_name,cleaner0,2023-07-05T10:42:18Z,SO:","id":"302"},{"span":{"begin":19780,"end":19794},"obj":"evidence,DUMMY:,melaniev@ebi.ac.uk,2023-07-06T15:26:33Z","id":"894"},{"span":{"begin":19850,"end":19854},"obj":"0.91976935,complex_assembly,cleaner0,2023-07-05T12:07:53Z,GO:","id":"304"},{"span":{"begin":19876,"end":19879},"obj":"0.9860378,residue_name,cleaner0,2023-07-05T10:42:35Z,SO:","id":"305"},{"span":{"begin":19884,"end":19887},"obj":"0.99085087,residue_name,cleaner0,2023-07-05T10:42:39Z,SO:","id":"306"},{"span":{"begin":19951,"end":19954},"obj":"0.9699916,chemical,cleaner0,2023-07-05T12:14:14Z,CHEBI:","id":"307"},{"span":{"begin":20086,"end":20110},"obj":"0.9950683,evidence,cleaner0,2023-07-05T12:21:30Z,DUMMY:","id":"308"},{"span":{"begin":20185,"end":20188},"obj":"0.97884846,residue_name,cleaner0,2023-07-05T10:42:43Z,SO:","id":"310"},{"span":{"begin":20202,"end":20208},"obj":"0.9714825,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"311"},{"span":{"begin":20305,"end":20321},"obj":"0.99046993,evidence,cleaner0,2023-07-05T12:21:33Z,DUMMY:","id":"312"},{"span":{"begin":20424,"end":20427},"obj":"0.98477256,residue_name,cleaner0,2023-07-05T12:07:11Z,SO:","id":"313"},{"span":{"begin":20432,"end":20435},"obj":"0.98784196,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"314"},{"span":{"begin":20463,"end":20469},"obj":"0.7084436,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"315"},{"span":{"begin":20715,"end":20721},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T10:43:57Z","id":"798"},{"span":{"begin":20793,"end":20796},"obj":"0.9841786,residue_name,cleaner0,2023-07-05T12:07:17Z,SO:","id":"317"},{"span":{"begin":20811,"end":20814},"obj":"0.9849412,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"318"},{"span":{"begin":21055,"end":21058},"obj":"0.993453,chemical,cleaner0,2023-07-05T12:14:18Z,CHEBI:","id":"319"},{"span":{"begin":21109,"end":21117},"obj":"0.9969453,protein_state,cleaner0,2023-07-05T12:31:56Z,DUMMY:","id":"320"},{"span":{"begin":21118,"end":21121},"obj":"0.98660594,residue_name,cleaner0,2023-07-05T12:07:19Z,SO:","id":"321"},{"span":{"begin":21147,"end":21150},"obj":"0.98063844,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"322"},{"span":{"begin":21189,"end":21192},"obj":"0.99770075,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"323"},{"span":{"begin":21216,"end":21232},"obj":"0.83079296,evidence,cleaner0,2023-07-05T10:43:12Z,DUMMY:","id":"324"},{"span":{"begin":21262,"end":21270},"obj":"0.9937124,complex_assembly,cleaner0,2023-07-05T10:38:43Z,GO:","id":"325"},{"span":{"begin":21285,"end":21305},"obj":"0.9262293,experimental_method,cleaner0,2023-07-05T12:16:54Z,MESH:","id":"326"},{"span":{"begin":21306,"end":21329},"obj":"0.9843981,evidence,cleaner0,2023-07-05T10:43:15Z,DUMMY:","id":"327"},{"span":{"begin":21360,"end":21363},"obj":"0.99798906,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"328"},{"span":{"begin":21364,"end":21392},"obj":"0.8665679,evidence,cleaner0,2023-07-05T10:43:17Z,DUMMY:","id":"329"},{"span":{"begin":21577,"end":21580},"obj":"0.9983839,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"330"},{"span":{"begin":21657,"end":21663},"obj":"0.88909703,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"331"},{"span":{"begin":21696,"end":21699},"obj":"0.9981768,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"332"},{"span":{"begin":21717,"end":21720},"obj":"0.9861231,residue_name,cleaner0,2023-07-05T12:07:24Z,SO:","id":"333"},{"span":{"begin":21725,"end":21728},"obj":"0.9858482,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"334"},{"span":{"begin":21844,"end":21847},"obj":"0.9861166,residue_name,cleaner0,2023-07-05T10:42:44Z,SO:","id":"335"},{"span":{"begin":21864,"end":21868},"obj":"0.97520036,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"336"},{"span":{"begin":21965,"end":21968},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"694"},{"span":{"begin":22063,"end":22067},"obj":"0.9912158,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"337"},{"span":{"begin":22068,"end":22072},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"824"},{"span":{"begin":22079,"end":22082},"obj":"0.989652,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"338"},{"span":{"begin":22091,"end":22094},"obj":"0.98959446,residue_name,cleaner0,2023-07-05T12:34:05Z,SO:","id":"339"},{"span":{"begin":22116,"end":22123},"obj":"0.9787446,oligomeric_state,cleaner0,2023-07-05T10:43:50Z,DUMMY:","id":"340"},{"span":{"begin":22235,"end":22238},"obj":"0.9698287,residue_name,cleaner0,2023-07-05T12:34:09Z,SO:","id":"341"},{"span":{"begin":22245,"end":22248},"obj":"0.95125747,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"342"},{"span":{"begin":22258,"end":22264},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T10:43:57Z","id":"799"},{"span":{"begin":22435,"end":22443},"obj":"0.9959565,protein_state,cleaner0,2023-07-05T10:44:09Z,DUMMY:","id":"344"},{"span":{"begin":22448,"end":22453},"obj":"0.9911316,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"345"},{"span":{"begin":22454,"end":22458},"obj":"0.99624735,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"346"},{"span":{"begin":22573,"end":22577},"obj":"0.8192733,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"347"},{"span":{"begin":22603,"end":22625},"obj":"0.99758184,site,cleaner0,2023-07-05T10:43:27Z,SO:","id":"348"},{"span":{"begin":22667,"end":22675},"obj":"0.9959115,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"349"},{"span":{"begin":22676,"end":22680},"obj":"0.99391973,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"350"},{"span":{"begin":22719,"end":22739},"obj":"0.9969743,site,cleaner0,2023-07-05T10:43:29Z,SO:","id":"351"},{"span":{"begin":22779,"end":22784},"obj":"0.9965004,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"352"},{"span":{"begin":22785,"end":22789},"obj":"0.9917121,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"353"},{"span":{"begin":22879,"end":22884},"obj":"0.99910825,residue_name_number,cleaner0,2023-07-05T10:44:41Z,DUMMY:","id":"354"},{"span":{"begin":22886,"end":22891},"obj":"0.99910176,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"355"},{"span":{"begin":22896,"end":22901},"obj":"0.99909663,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"356"},{"span":{"begin":22919,"end":22922},"obj":"0.9915868,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"357"},{"span":{"begin":22958,"end":22962},"obj":"0.7789828,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"358"},{"span":{"begin":22963,"end":22967},"obj":"0.4135374,structure_element,cleaner0,2023-07-05T11:58:12Z,SO:","id":"359"},{"span":{"begin":22968,"end":22976},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:54Z","id":"883"},{"span":{"begin":23003,"end":23018},"obj":"0.9754962,evidence,cleaner0,2023-07-05T12:21:41Z,DUMMY:","id":"360"},{"span":{"begin":23041,"end":23067},"obj":"0.9385281,experimental_method,cleaner0,2023-07-05T10:44:16Z,MESH:","id":"361"},{"span":{"begin":23101,"end":23117},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:17:44Z","id":"888"},{"span":{"begin":23180,"end":23193},"obj":"0.9535044,evidence,cleaner0,2023-07-05T12:21:49Z,DUMMY:","id":"363"},{"span":{"begin":23213,"end":23218},"obj":"0.99416935,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"364"},{"span":{"begin":23223,"end":23231},"obj":"0.98500234,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"365"},{"span":{"begin":23287,"end":23293},"obj":"0.95402676,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"366"},{"span":{"begin":23306,"end":23311},"obj":"0.9990717,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"367"},{"span":{"begin":23315,"end":23324},"obj":"0.99607676,protein_state,cleaner0,2023-07-05T12:32:01Z,DUMMY:","id":"368"},{"span":{"begin":23339,"end":23347},"obj":"0.9939761,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"369"},{"span":{"begin":23348,"end":23352},"obj":"0.94174945,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"370"},{"span":{"begin":23451,"end":23455},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:07:58Z","id":"741"},{"span":{"begin":23456,"end":23460},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"827"},{"span":{"begin":23461,"end":23468},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:42Z","id":"880"},{"span":{"begin":23474,"end":23479},"obj":"0.9990544,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"372"},{"span":{"begin":23567,"end":23569},"obj":"0.98224247,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"373"},{"span":{"begin":23570,"end":23573},"obj":"0.996082,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"374"},{"span":{"begin":23597,"end":23602},"obj":"0.99904436,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"375"},{"span":{"begin":23610,"end":23627},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:00:19Z","id":"833"},{"span":{"begin":23628,"end":23637},"obj":"0.9971259,evidence,cleaner0,2023-07-05T12:21:52Z,DUMMY:","id":"378"},{"span":{"begin":23692,"end":23694},"obj":"0.43124557,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"379"},{"span":{"begin":23722,"end":23741},"obj":"site,SO:,cleaner0,2023-07-05T10:45:21Z","id":"806"},{"span":{"begin":23751,"end":23756},"obj":"0.99898416,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"383"},{"span":{"begin":23760,"end":23762},"obj":"0.5508266,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"384"},{"span":{"begin":23790,"end":23797},"obj":"0.8253004,evidence,cleaner0,2023-07-05T12:21:57Z,DUMMY:","id":"385"},{"span":{"begin":23815,"end":23818},"obj":"0.59934,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"386"},{"span":{"begin":23855,"end":23860},"obj":"0.9990036,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"387"},{"span":{"begin":23870,"end":23875},"obj":"0.99900705,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"388"},{"span":{"begin":23913,"end":23916},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"697"},{"span":{"begin":24004,"end":24009},"obj":"0.9987974,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"390"},{"span":{"begin":24054,"end":24059},"obj":"0.9982838,chemical,cleaner0,2023-07-05T12:14:22Z,CHEBI:","id":"391"},{"span":{"begin":24072,"end":24095},"obj":"site,SO:,cleaner0,2023-07-05T10:45:45Z","id":"807"},{"span":{"begin":24167,"end":24172},"obj":"0.99884206,residue_name_number,cleaner0,2023-07-05T10:45:53Z,DUMMY:","id":"394"},{"span":{"begin":24289,"end":24298},"obj":"0.99792117,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:26:50Z,SO:","id":"395"},{"span":{"begin":24349,"end":24359},"obj":"0.99548495,protein_type,cleaner0,2023-07-05T12:27:28Z,MESH:","id":"396"},{"span":{"begin":24368,"end":24377},"obj":"0.996011,evidence,cleaner0,2023-07-05T12:22:00Z,DUMMY:","id":"397"},{"span":{"begin":24448,"end":24463},"obj":"0.98592824,evidence,cleaner0,2023-07-05T12:22:03Z,DUMMY:","id":"398"},{"span":{"begin":24472,"end":24480},"obj":"0.995103,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"399"},{"span":{"begin":24481,"end":24486},"obj":"0.98567003,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"400"},{"span":{"begin":24487,"end":24492},"obj":"0.9971819,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"401"},{"span":{"begin":24543,"end":24548},"obj":"0.99736005,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"402"},{"span":{"begin":24676,"end":24679},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"699"},{"span":{"begin":24701,"end":24706},"obj":"0.6723021,chemical,cleaner0,2023-07-05T12:14:46Z,CHEBI:","id":"404"},{"span":{"begin":24797,"end":24807},"obj":"0.99513197,protein_state,cleaner0,2023-07-05T12:32:17Z,DUMMY:","id":"405"},{"span":{"begin":24808,"end":24811},"obj":"0.97005063,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"406"},{"span":{"begin":24817,"end":24840},"obj":"0.99278337,evidence,cleaner0,2023-07-05T12:22:06Z,DUMMY:","id":"407"},{"span":{"begin":24886,"end":24891},"obj":"0.99656266,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"408"},{"span":{"begin":24896,"end":24904},"obj":"0.9923832,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"409"},{"span":{"begin":24905,"end":24909},"obj":"0.23798376,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"410"},{"span":{"begin":24919,"end":24924},"obj":"0.99764353,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"411"},{"span":{"begin":24982,"end":24985},"obj":"0.4976674,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"412"},{"span":{"begin":25064,"end":25069},"obj":"0.9930019,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"414"},{"span":{"begin":25119,"end":25122},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"700"},{"span":{"begin":25219,"end":25224},"obj":"0.99905163,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"416"},{"span":{"begin":25244,"end":25246},"obj":"0.9022129,residue_name_number,cleaner0,2023-07-05T10:52:10Z,DUMMY:","id":"417"},{"span":{"begin":25266,"end":25293},"obj":"site,SO:,cleaner0,2023-07-05T10:46:58Z","id":"808"},{"span":{"begin":25343,"end":25349},"obj":"0.9752722,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"421"},{"span":{"begin":25370,"end":25375},"obj":"0.999049,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"422"},{"span":{"begin":25418,"end":25421},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:21Z","id":"702"},{"span":{"begin":25443,"end":25448},"obj":"0.99901557,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"423"},{"span":{"begin":25531,"end":25536},"obj":"0.99905235,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"424"},{"span":{"begin":25578,"end":25583},"obj":"0.99905616,residue_name_number,cleaner0,2023-07-05T10:51:41Z,DUMMY:","id":"425"},{"span":{"begin":25621,"end":25623},"obj":"0.54564714,residue_name_number,cleaner0,2023-07-05T10:52:23Z,DUMMY:","id":"426"},{"span":{"begin":25679,"end":25700},"obj":"0.99768096,site,cleaner0,2023-07-05T12:04:01Z,SO:","id":"427"},{"span":{"begin":25706,"end":25712},"obj":"0.9751469,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"428"},{"span":{"begin":25717,"end":25722},"obj":"0.99891317,residue_name_number,cleaner0,2023-07-05T10:51:42Z,DUMMY:","id":"429"},{"span":{"begin":25782,"end":25784},"obj":"0.7176499,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"430"},{"span":{"begin":25818,"end":25823},"obj":"0.9988103,residue_name_number,cleaner0,2023-07-05T10:51:42Z,DUMMY:","id":"431"},{"span":{"begin":25850,"end":25855},"obj":"0.99893457,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"432"},{"span":{"begin":25860,"end":25865},"obj":"0.9989254,residue_name_number,cleaner0,2023-07-05T10:51:42Z,DUMMY:","id":"433"},{"span":{"begin":25946,"end":25948},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T10:28:30Z","id":"754"},{"span":{"begin":26012,"end":26020},"obj":"0.9915398,complex_assembly,cleaner0,2023-07-05T10:38:43Z,GO:","id":"434"},{"span":{"begin":26162,"end":26191},"obj":"0.9947875,evidence,cleaner0,2023-07-05T12:22:11Z,DUMMY:","id":"435"},{"span":{"begin":26236,"end":26242},"obj":"0.78213966,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"437"},{"span":{"begin":26362,"end":26364},"obj":"0.64419585,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"438"},{"span":{"begin":26539,"end":26591},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T10:54:22Z","id":"815"},{"span":{"begin":26645,"end":26652},"obj":"0.98756266,evidence,cleaner0,2023-07-05T12:22:22Z,DUMMY:","id":"441"},{"span":{"begin":26719,"end":26722},"obj":"0.9878491,chemical,cleaner0,2023-07-05T10:26:21Z,CHEBI:","id":"442"},{"span":{"begin":26782,"end":26791},"obj":"0.9950579,protein_state,cleaner0,2023-07-05T12:32:29Z,DUMMY:","id":"443"},{"span":{"begin":26796,"end":26804},"obj":"0.9650826,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"444"},{"span":{"begin":26805,"end":26809},"obj":"0.45595852,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"445"},{"span":{"begin":26839,"end":26841},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T10:28:30Z","id":"755"},{"span":{"begin":26866,"end":26869},"obj":"0.97414523,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"446"},{"span":{"begin":26905,"end":26924},"obj":"0.7477479,protein_state,cleaner0,2023-07-05T12:32:46Z,DUMMY:","id":"447"},{"span":{"begin":27044,"end":27061},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:17:38Z","id":"889"},{"span":{"begin":27069,"end":27079},"obj":"0.99410534,complex_assembly,cleaner0,2023-07-05T12:31:03Z,GO:","id":"449"},{"span":{"begin":27088,"end":27091},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"767"},{"span":{"begin":27181,"end":27216},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T10:54:03Z","id":"814"},{"span":{"begin":27265,"end":27268},"obj":"0.9961176,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"455"},{"span":{"begin":27299,"end":27317},"obj":"0.9903002,evidence,cleaner0,2023-07-05T12:22:26Z,DUMMY:","id":"456"},{"span":{"begin":27331,"end":27334},"obj":"0.99770594,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"457"},{"span":{"begin":27352,"end":27360},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:54Z","id":"884"},{"span":{"begin":27362,"end":27365},"obj":"0.9490286,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"458"},{"span":{"begin":27443,"end":27446},"obj":"0.98482555,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"459"},{"span":{"begin":27528,"end":27530},"obj":"0.6900701,residue_name_number,cleaner0,2023-07-05T10:53:16Z,DUMMY:","id":"460"},{"span":{"begin":27531,"end":27540},"obj":"0.5953651,chemical,cleaner0,2023-07-05T12:14:51Z,CHEBI:","id":"461"},{"span":{"begin":27569,"end":27575},"obj":"0.9657221,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"462"},{"span":{"begin":27601,"end":27603},"obj":"0.48627108,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"463"},{"span":{"begin":27605,"end":27607},"obj":"0.43101338,residue_name_number,cleaner0,2023-07-05T10:54:30Z,DUMMY:","id":"464"},{"span":{"begin":27612,"end":27614},"obj":"0.69601625,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"465"},{"span":{"begin":27646,"end":27648},"obj":"0.97514343,residue_name_number,cleaner0,2023-07-05T10:53:17Z,DUMMY:","id":"466"},{"span":{"begin":27740,"end":27744},"obj":"0.8990134,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"468"},{"span":{"begin":27753,"end":27758},"obj":"0.9884177,chemical,cleaner0,2023-07-05T12:15:01Z,CHEBI:","id":"469"},{"span":{"begin":27815,"end":27823},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:54Z","id":"885"},{"span":{"begin":27837,"end":27842},"obj":"0.99823785,residue_name_number,cleaner0,2023-07-05T10:45:53Z,DUMMY:","id":"470"},{"span":{"begin":27885,"end":27893},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:54Z","id":"886"},{"span":{"begin":27911,"end":27913},"obj":"0.9919527,residue_name_number,cleaner0,2023-07-05T10:53:17Z,DUMMY:","id":"471"},{"span":{"begin":27914,"end":27920},"obj":"0.7579533,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"472"},{"span":{"begin":28006,"end":28009},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:22Z","id":"706"},{"span":{"begin":28019,"end":28025},"obj":"0.66986865,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"473"},{"span":{"begin":28081,"end":28084},"obj":"0.9113056,residue_name,cleaner0,2023-07-05T10:42:44Z,SO:","id":"474"},{"span":{"begin":28264,"end":28267},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:22Z","id":"707"},{"span":{"begin":28295,"end":28312},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T10:53:41Z","id":"813"},{"span":{"begin":28414,"end":28417},"obj":"0.86929446,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"478"},{"span":{"begin":28527,"end":28530},"obj":"0.99296665,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"479"},{"span":{"begin":28548,"end":28552},"obj":"0.98447126,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"480"},{"span":{"begin":28646,"end":28651},"obj":"0.9987546,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"481"},{"span":{"begin":28656,"end":28661},"obj":"0.99878544,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"482"},{"span":{"begin":28679,"end":28682},"obj":"0.99426657,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"483"},{"span":{"begin":28719,"end":28726},"obj":"0.97147286,chemical,cleaner0,2023-07-05T12:15:09Z,CHEBI:","id":"484"},{"span":{"begin":28734,"end":28736},"obj":"0.92182314,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"485"},{"span":{"begin":28741,"end":28743},"obj":"0.95111024,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"486"},{"span":{"begin":28783,"end":28788},"obj":"0.998728,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"487"},{"span":{"begin":28790,"end":28795},"obj":"0.99873954,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"488"},{"span":{"begin":28835,"end":28843},"obj":"0.9912282,protein_state,cleaner0,2023-07-05T12:32:51Z,DUMMY:","id":"489"},{"span":{"begin":28844,"end":28847},"obj":"0.9868809,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"490"},{"span":{"begin":28900,"end":28943},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:18:19Z","id":"890"},{"span":{"begin":28993,"end":28995},"obj":"0.7989518,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"493"},{"span":{"begin":29024,"end":29028},"obj":"0.98919463,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"495"},{"span":{"begin":29047,"end":29049},"obj":"0.78089446,residue_name_number,cleaner0,2023-07-05T10:54:30Z,DUMMY:","id":"496"},{"span":{"begin":29054,"end":29056},"obj":"0.8507618,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"497"},{"span":{"begin":29092,"end":29112},"obj":"0.98744667,evidence,cleaner0,2023-07-05T12:22:33Z,DUMMY:","id":"498"},{"span":{"begin":29116,"end":29120},"obj":"0.99020547,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"499"},{"span":{"begin":29122,"end":29125},"obj":"0.99115556,evidence,cleaner0,2023-07-05T12:22:36Z,DUMMY:","id":"500"},{"span":{"begin":29147,"end":29152},"obj":"0.99872905,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"501"},{"span":{"begin":29157,"end":29162},"obj":"0.998755,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"502"},{"span":{"begin":29253,"end":29259},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T10:43:57Z","id":"802"},{"span":{"begin":29261,"end":29271},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:23:06Z","id":"895"},{"span":{"begin":29389,"end":29394},"obj":"0.9987036,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"504"},{"span":{"begin":29419,"end":29428},"obj":"0.9961844,protein_state,cleaner0,2023-07-05T12:32:54Z,DUMMY:","id":"505"},{"span":{"begin":29429,"end":29433},"obj":"0.9805304,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"506"},{"span":{"begin":29439,"end":29444},"obj":"0.9988978,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"507"},{"span":{"begin":29506,"end":29511},"obj":"0.9990214,residue_name_number,cleaner0,2023-07-05T10:55:21Z,DUMMY:","id":"508"},{"span":{"begin":29516,"end":29521},"obj":"0.99905556,residue_name_number,cleaner0,2023-07-05T10:55:26Z,DUMMY:","id":"509"},{"span":{"begin":29556,"end":29565},"obj":"0.95854217,protein_state,cleaner0,2023-07-05T12:32:59Z,DUMMY:","id":"510"},{"span":{"begin":29582,"end":29584},"obj":"0.996298,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"511"},{"span":{"begin":29666,"end":29671},"obj":"0.9989222,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"512"},{"span":{"begin":29675,"end":29684},"obj":"0.9960868,protein_state,cleaner0,2023-07-05T12:33:02Z,DUMMY:","id":"513"},{"span":{"begin":29685,"end":29689},"obj":"0.9864499,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"514"},{"span":{"begin":30039,"end":30047},"obj":"0.9948141,protein_state,cleaner0,2023-07-05T12:33:06Z,DUMMY:","id":"515"},{"span":{"begin":30048,"end":30051},"obj":"0.99623907,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"516"},{"span":{"begin":30096,"end":30101},"obj":"0.99894124,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"517"},{"span":{"begin":30196,"end":30201},"obj":"0.9985372,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"518"},{"span":{"begin":30242,"end":30264},"obj":"0.76363486,evidence,cleaner0,2023-07-05T12:23:16Z,DUMMY:","id":"519"},{"span":{"begin":30345,"end":30350},"obj":"0.9988926,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"520"},{"span":{"begin":30440,"end":30444},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:07:58Z","id":"745"},{"span":{"begin":30450,"end":30482},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:23:39Z","id":"896"},{"span":{"begin":30552,"end":30563},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:04:50Z","id":"838"},{"span":{"begin":30586,"end":30591},"obj":"0.99887615,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"523"},{"span":{"begin":30592,"end":30605},"obj":"0.97079873,site,cleaner0,2023-07-05T12:34:26Z,SO:","id":"524"},{"span":{"begin":30625,"end":30628},"obj":"0.8287284,residue_name,cleaner0,2023-07-05T12:34:16Z,SO:","id":"525"},{"span":{"begin":30652,"end":30655},"obj":"0.9915752,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"526"},{"span":{"begin":30669,"end":30674},"obj":"0.998931,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"527"},{"span":{"begin":30774,"end":30777},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T12:23:52Z","id":"897"},{"span":{"begin":30788,"end":30796},"obj":"0.59207654,evidence,cleaner0,2023-07-05T12:23:58Z,DUMMY:","id":"528"},{"span":{"begin":31112,"end":31119},"obj":"0.99064636,evidence,cleaner0,2023-07-05T12:24:02Z,DUMMY:","id":"530"},{"span":{"begin":31543,"end":31547},"obj":"0.86443084,protein_state,cleaner0,2023-07-05T12:33:09Z,DUMMY:","id":"533"},{"span":{"begin":31552,"end":31559},"obj":"0.7418846,protein_state,cleaner0,2023-07-05T12:33:11Z,DUMMY:","id":"534"},{"span":{"begin":31560,"end":31563},"obj":"0.9973688,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"535"},{"span":{"begin":31991,"end":32013},"obj":"0.99785984,site,cleaner0,2023-07-05T12:34:37Z,SO:","id":"537"},{"span":{"begin":32015,"end":32018},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:22Z","id":"712"},{"span":{"begin":32076,"end":32081},"obj":"0.999079,residue_name_number,cleaner0,2023-07-05T10:56:05Z,DUMMY:","id":"538"},{"span":{"begin":32083,"end":32088},"obj":"0.99905926,residue_name_number,cleaner0,2023-07-05T10:56:10Z,DUMMY:","id":"539"},{"span":{"begin":32093,"end":32098},"obj":"0.9990657,residue_name_number,cleaner0,2023-07-05T10:56:14Z,DUMMY:","id":"540"},{"span":{"begin":32154,"end":32158},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:07:58Z","id":"746"},{"span":{"begin":32159,"end":32164},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:05:25Z","id":"843"},{"span":{"begin":32268,"end":32273},"obj":"0.9930227,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"542"},{"span":{"begin":32278,"end":32286},"obj":"0.9660058,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"543"},{"span":{"begin":32287,"end":32291},"obj":"0.9864768,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"544"},{"span":{"begin":32328,"end":32332},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:07:58Z","id":"747"},{"span":{"begin":32333,"end":32337},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"828"},{"span":{"begin":32355,"end":32360},"obj":"0.9962069,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"546"},{"span":{"begin":32361,"end":32365},"obj":"0.9905319,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"547"},{"span":{"begin":32367,"end":32372},"obj":"0.9990728,residue_name_number,cleaner0,2023-07-05T10:56:14Z,DUMMY:","id":"548"},{"span":{"begin":32400,"end":32406},"obj":"0.99010086,residue_name,cleaner0,2023-07-05T10:56:37Z,SO:","id":"549"},{"span":{"begin":32425,"end":32433},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:54Z","id":"881"},{"span":{"begin":32446,"end":32454},"obj":"0.9937517,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"550"},{"span":{"begin":32455,"end":32459},"obj":"0.9874014,complex_assembly,cleaner0,2023-07-05T12:07:58Z,GO:","id":"551"},{"span":{"begin":32491,"end":32496},"obj":"0.999064,residue_name_number,cleaner0,2023-07-05T10:56:14Z,DUMMY:","id":"552"},{"span":{"begin":32541,"end":32547},"obj":"0.9762691,chemical,cleaner0,2023-07-05T12:15:18Z,CHEBI:","id":"553"},{"span":{"begin":32556,"end":32563},"obj":"structure_element,SO:,cleaner0,2023-07-05T12:15:42Z","id":"879"},{"span":{"begin":32756,"end":32774},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T12:18:42Z","id":"891"},{"span":{"begin":33054,"end":33056},"obj":"0.74215937,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"555"},{"span":{"begin":33080,"end":33083},"obj":"0.95398015,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"556"},{"span":{"begin":33095,"end":33099},"obj":"0.9726016,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"557"},{"span":{"begin":33153,"end":33157},"obj":"0.8587137,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"558"},{"span":{"begin":33186,"end":33191},"obj":"0.9990075,residue_name_number,cleaner0,2023-07-05T10:51:42Z,DUMMY:","id":"559"},{"span":{"begin":33196,"end":33201},"obj":"0.99903595,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"560"},{"span":{"begin":33227,"end":33229},"obj":"0.65299237,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"561"},{"span":{"begin":33234,"end":33236},"obj":"0.5014381,residue_name_number,cleaner0,2023-07-05T10:52:11Z,DUMMY:","id":"562"},{"span":{"begin":33304,"end":33307},"obj":"0.98193204,residue_name,cleaner0,2023-07-05T10:56:31Z,SO:","id":"563"},{"span":{"begin":33530,"end":33534},"obj":"0.9829249,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"565"},{"span":{"begin":33536,"end":33542},"obj":"0.6355414,evidence,cleaner0,2023-07-05T10:43:57Z,DUMMY:","id":"566"},{"span":{"begin":33584,"end":33587},"obj":"0.97448653,residue_name,cleaner0,2023-07-05T10:56:34Z,SO:","id":"567"},{"span":{"begin":33609,"end":33613},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"829"},{"span":{"begin":33642,"end":33646},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"830"},{"span":{"begin":33725,"end":33749},"obj":"0.9927671,evidence,cleaner0,2023-07-05T12:24:08Z,DUMMY:","id":"568"},{"span":{"begin":33771,"end":33774},"obj":"0.56909597,evidence,cleaner0,2023-07-05T12:24:17Z,DUMMY:","id":"569"},{"span":{"begin":33806,"end":33809},"obj":"0.9574091,residue_name,cleaner0,2023-07-05T10:56:40Z,SO:","id":"570"},{"span":{"begin":33841,"end":33844},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:26:22Z","id":"713"},{"span":{"begin":33940,"end":33962},"obj":"0.99812096,site,cleaner0,2023-07-05T12:34:42Z,SO:","id":"571"},{"span":{"begin":33974,"end":33978},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:07:59Z","id":"748"},{"span":{"begin":33979,"end":33983},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"831"},{"span":{"begin":33998,"end":34003},"obj":"0.9991387,residue_name_number,cleaner0,2023-07-05T12:00:25Z,DUMMY:","id":"573"},{"span":{"begin":34031,"end":34033},"obj":"0.48927557,residue_name_number,cleaner0,2023-07-05T10:52:24Z,DUMMY:","id":"574"},{"span":{"begin":34052,"end":34074},"obj":"0.9979715,site,cleaner0,2023-07-05T12:34:45Z,SO:","id":"575"},{"span":{"begin":34112,"end":34130},"obj":"0.9856348,evidence,cleaner0,2023-07-05T12:24:21Z,DUMMY:","id":"576"},{"span":{"begin":34139,"end":34148},"obj":"0.99598575,protein_state,cleaner0,2023-07-05T12:33:16Z,DUMMY:","id":"577"},{"span":{"begin":34153,"end":34161},"obj":"0.9913564,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"578"},{"span":{"begin":34162,"end":34166},"obj":"0.9880157,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"579"},{"span":{"begin":34392,"end":34403},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:06:23Z","id":"874"},{"span":{"begin":34408,"end":34419},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:06:42Z","id":"876"},{"span":{"begin":34486,"end":34495},"obj":"0.99518895,protein_state,cleaner0,2023-07-05T12:33:22Z,DUMMY:","id":"582"},{"span":{"begin":34596,"end":34598},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T10:28:30Z","id":"756"},{"span":{"begin":34693,"end":34708},"obj":"0.9768747,protein_type,cleaner0,2023-07-05T12:27:33Z,MESH:","id":"584"},{"span":{"begin":34717,"end":34739},"obj":"0.992797,site,cleaner0,2023-07-05T12:35:08Z,SO:","id":"585"},{"span":{"begin":34956,"end":34959},"obj":"0.98401004,chemical,cleaner0,2023-07-05T10:36:34Z,CHEBI:","id":"587"},{"span":{"begin":34964,"end":34967},"obj":"0.98465955,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"588"},{"span":{"begin":35124,"end":35127},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"772"},{"span":{"begin":35152,"end":35181},"obj":"0.99563825,site,cleaner0,2023-07-05T12:35:12Z,SO:","id":"589"},{"span":{"begin":35299,"end":35302},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"774"},{"span":{"begin":35369,"end":35371},"obj":"0.62370133,experimental_method,cleaner0,2023-07-05T10:28:30Z,MESH:","id":"591"},{"span":{"begin":35487,"end":35498},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T12:04:56Z","id":"842"},{"span":{"begin":35606,"end":35619},"obj":"0.53609496,complex_assembly,cleaner0,2023-07-05T12:13:58Z,GO:","id":"593"},{"span":{"begin":35760,"end":35780},"obj":"0.9972315,site,cleaner0,2023-07-05T12:34:49Z,SO:","id":"594"},{"span":{"begin":35875,"end":35880},"obj":"protein_state,DUMMY:,cleaner0,2023-07-05T10:43:39Z","id":"794"},{"span":{"begin":35900,"end":35903},"obj":"0.9973586,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"595"},{"span":{"begin":35984,"end":35992},"obj":"0.98719054,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"596"},{"span":{"begin":35993,"end":35997},"obj":"0.6919731,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"597"},{"span":{"begin":36055,"end":36077},"obj":"0.9982071,site,cleaner0,2023-07-05T12:34:52Z,SO:","id":"599"},{"span":{"begin":36079,"end":36084},"obj":"0.9990889,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"600"},{"span":{"begin":36086,"end":36091},"obj":"0.99907863,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"601"},{"span":{"begin":36093,"end":36098},"obj":"0.9990723,residue_name_number,cleaner0,2023-07-05T10:44:50Z,DUMMY:","id":"602"},{"span":{"begin":36317,"end":36321},"obj":"0.87554866,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"603"},{"span":{"begin":36331,"end":36334},"obj":"0.99667054,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"604"},{"span":{"begin":36452,"end":36455},"obj":"0.9962542,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"605"},{"span":{"begin":36513,"end":36516},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T10:36:34Z","id":"775"},{"span":{"begin":41496,"end":41513},"obj":"complex_assembly,GO:,cleaner0,2023-07-05T10:58:32Z","id":"817"},{"span":{"begin":41579,"end":41584},"obj":"0.99105424,protein_state,cleaner0,2023-07-05T10:43:39Z,DUMMY:","id":"609"},{"span":{"begin":41585,"end":41595},"obj":"0.9840899,chemical,cleaner0,2023-07-05T12:16:01Z,CHEBI:","id":"610"},{"span":{"begin":41641,"end":41644},"obj":"0.9981456,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"611"},{"span":{"begin":41715,"end":41721},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:12:10Z","id":"877"},{"span":{"begin":41729,"end":41737},"obj":"0.9932771,complex_assembly,cleaner0,2023-07-05T10:38:44Z,GO:","id":"615"},{"span":{"begin":41746,"end":41753},"obj":"0.9954058,evidence,cleaner0,2023-07-05T12:24:38Z,DUMMY:","id":"616"},{"span":{"begin":41835,"end":41851},"obj":"0.7991534,evidence,cleaner0,2023-07-05T12:24:42Z,DUMMY:","id":"617"},{"span":{"begin":41873,"end":41881},"obj":"0.99266356,complex_assembly,cleaner0,2023-07-05T10:38:44Z,GO:","id":"618"},{"span":{"begin":41882,"end":41891},"obj":"0.98201466,evidence,cleaner0,2023-07-05T12:24:45Z,DUMMY:","id":"619"},{"span":{"begin":41906,"end":41920},"obj":"0.9719779,evidence,cleaner0,2023-07-05T12:24:49Z,DUMMY:","id":"620"},{"span":{"begin":41922,"end":41945},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:25:15Z","id":"898"},{"span":{"begin":42115,"end":42129},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:13:13Z","id":"878"},{"span":{"begin":42183,"end":42186},"obj":"0.96845776,evidence,cleaner0,2023-07-05T12:25:20Z,DUMMY:","id":"626"},{"span":{"begin":42188,"end":42213},"obj":"0.98903877,evidence,cleaner0,2023-07-05T12:25:23Z,DUMMY:","id":"627"},{"span":{"begin":42313,"end":42317},"obj":"0.5221266,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"628"},{"span":{"begin":42350,"end":42364},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:25:43Z","id":"899"},{"span":{"begin":42392,"end":42396},"obj":"0.7290474,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"630"},{"span":{"begin":42460,"end":42507},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T12:26:14Z","id":"900"},{"span":{"begin":42630,"end":42648},"obj":"0.9945644,evidence,cleaner0,2023-07-05T12:26:18Z,DUMMY:","id":"638"},{"span":{"begin":42672,"end":42677},"obj":"0.9991478,residue_name_number,cleaner0,2023-07-05T10:44:51Z,DUMMY:","id":"639"},{"span":{"begin":42721,"end":42747},"obj":"0.8718893,evidence,cleaner0,2023-07-05T12:26:29Z,DUMMY:","id":"641"},{"span":{"begin":42896,"end":42899},"obj":"0.997905,chemical,cleaner0,2023-07-05T10:26:22Z,CHEBI:","id":"643"},{"span":{"begin":42900,"end":42907},"obj":"0.9303295,evidence,cleaner0,2023-07-05T12:26:36Z,DUMMY:","id":"644"},{"span":{"begin":42939,"end":42945},"obj":"0.89408696,evidence,cleaner0,2023-07-05T12:26:41Z,DUMMY:","id":"645"},{"span":{"begin":42991,"end":42994},"obj":"0.9847978,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:26:50Z,SO:","id":"646"},{"span":{"begin":43002,"end":43005},"obj":"0.97973424,residue_name,cleaner0,2023-07-05T10:42:19Z,SO:","id":"647"},{"span":{"begin":43026,"end":43034},"obj":"0.9919364,complex_assembly,cleaner0,2023-07-05T10:38:44Z,GO:","id":"648"},{"span":{"begin":43137,"end":43142},"obj":"0.98781407,protein_state,cleaner0,2023-07-05T10:43:40Z,DUMMY:","id":"649"},{"span":{"begin":43147,"end":43155},"obj":"0.94484127,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"650"},{"span":{"begin":43248,"end":43252},"obj":"0.72071564,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"651"},{"span":{"begin":43253,"end":43257},"obj":"structure_element,SO:,cleaner0,2023-07-05T11:58:12Z","id":"832"},{"span":{"begin":43289,"end":43295},"obj":"0.93198663,evidence,cleaner0,2023-07-05T12:26:44Z,DUMMY:","id":"652"},{"span":{"begin":43398,"end":43404},"obj":"0.7863771,evidence,cleaner0,2023-07-05T12:26:53Z,DUMMY:","id":"653"},{"span":{"begin":43426,"end":43431},"obj":"0.99909854,residue_name_number,cleaner0,2023-07-05T10:44:42Z,DUMMY:","id":"655"},{"span":{"begin":43440,"end":43445},"obj":"0.9990896,residue_name_number,cleaner0,2023-07-05T10:44:46Z,DUMMY:","id":"656"},{"span":{"begin":43454,"end":43459},"obj":"0.9990759,residue_name_number,cleaner0,2023-07-05T10:44:51Z,DUMMY:","id":"657"},{"span":{"begin":43469,"end":43474},"obj":"0.9990729,residue_name_number,cleaner0,2023-07-05T10:44:51Z,DUMMY:","id":"658"},{"span":{"begin":43484,"end":43489},"obj":"0.9990627,residue_name_number,cleaner0,2023-07-05T10:51:36Z,DUMMY:","id":"659"},{"span":{"begin":43501,"end":43506},"obj":"0.9990792,residue_name_number,cleaner0,2023-07-05T10:51:42Z,DUMMY:","id":"660"},{"span":{"begin":43584,"end":43589},"obj":"0.9931265,protein_state,cleaner0,2023-07-05T10:43:40Z,DUMMY:","id":"661"},{"span":{"begin":43590,"end":43598},"obj":"0.9739872,protein_state,cleaner0,2023-07-05T10:44:10Z,DUMMY:","id":"662"},{"span":{"begin":43600,"end":43621},"obj":"0.9972383,site,cleaner0,2023-07-05T12:34:57Z,SO:","id":"663"},{"span":{"begin":43649,"end":43653},"obj":"0.95457613,complex_assembly,cleaner0,2023-07-05T12:07:59Z,GO:","id":"664"},{"span":{"begin":43672,"end":43718},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T10:59:56Z","id":"818"}],"relations":[]}]

annotated_BioC_JSON/PMC4871749_ann.json

@@ -0,0 +1 @@
+[{"sourceid":"4871749","sourcedb":"","project":"","target":"","text":"The Taf14 YEATS domain is a reader of histone crotonylation The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. This epigenetic PTM is widespread and enriched at active gene promoters and potentially enhancers. The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. We found that H3K9cr is present in yeast and is dynamically regulated. To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group. In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). Binding of the Taf14 YEATS domain to H3K9cr is robust. The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex. To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). We concluded that H3K9cr is the preferred target of this domain. From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine. In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare. ONLINE METHODS Protein expression and purification The Taf14 YEATS constructs (residues 1–132 or 1–137) were expressed in E. coli BL21 (DE3) RIL in either Luria Broth or M19 minimal media supplemented with 15NH4Cl and purified as N-terminal GST fusion proteins. Cells were harvested by centrifugation and resuspended in 50 mM HEPES (pH 7.5) supplemented with 150 mM NaCl and 1 mM TCEP. Cells are lysed by freeze-thaw followed by sonication. Proteins were purified on glutathione Sepharose 4B beads and the GST tag was cleaved with PreScission protease. X-ray data collection and structure determination Taf14 YEATS (residues 1–137) was concentrated to 9 mg/mL in 25 mM MES (pH 6.5) and incubated with 2 molar equivalence of the H3K9cr5-13 at RT for 30 mins prior to crystallization. Crystals were obtain via sitting drop diffusion method at 18°C by mixing 800 nL of protein/peptide solution with 800 nL of well solution composed of 44% PEG600 (v/v) and 0.2 M citric acid (pH 6.0). X-ray diffraction data was collected at a wavelength of 1.54 Å at 100 K from a single crystal on the UC Denver Biophysical Core home source composed of a Rigaku Micromax 007 high frequency microfocus X-ray generator with a Pilatus 200K 2D area detector. HKL3000 was used for indexing, scaling, and data reduction. Solution was solved via molecular replacement with Phaser using the Taf14 YEATS domain (PDB 5D7E) as search model with waters, ligands, and peptide removed. Phenix was used for refinement of structure and waters were manually placed by inception of difference maps in Coot. Ramachandran plot indicates good stereochemistry of the three-dimensional structure with 100% of all residues falling within the favored (98%) and allowed (2%) regions. The crystallographic statistics are shown in Supplementary Table 1. NMR spectroscopy NMR spectroscopy was carried out on a Varian INOVA 600 MHz spectrometer outfitted with a cryogenic probe. Chemical shift perturbation (CSP) analysis was performed using uniformly 15N-labeled Taf14 (1–132). 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the Taf14 YEATS domain were collected in the presence of increasing concentrations of either H3K9cr5-13, H3K9ac5-13, H3K9cr1-20, H3K9ac1-20 H3K9pr1-20, H3K9bu1-20 or free Kcr in PBS buffer pH 6.8, 8% D2O. Fluorescence binding assays Tryptophan fluorescence measurements were performed on a Fluorolog spectrofluorometer at room temperature as described. The samples containing 2 μM of Taf14 YEATS in PBS (pH 7.4) and increasing concentrations of H3K9cr5-13 were excited at 295 nm. Emission spectra were recorded from 310 to 340 nm with a 1 nm step size and a 0.5 sec integration time. The Kd value was determined using a nonlinear least-squares analysis and the equation:   where [L] is the concentration of the peptide, [P] is the concentration of the protein, ΔI is the observed change of signal intensity, and ΔImax is the difference in signal intensity of the free and bound states. The Kd values were averaged over 3 separate experiments, with error calculated as the standard deviation (SD). Peptide pull-downs YEATS domains in pGEX vectors were expressed in SoluBL21 cells (Amsbio) by induction with 1 mM IPTG at 16–18°C overnight with shaking. Cells were lysed by freeze-thaw and sonication then purified over glutathione agarose (Pierce) in a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol (v/v) and 1 mM dithiothreitol (DTT). Peptide pull-downs were performed essentially as described except that the assay buffer contained 50 mM Tris pH 8.0, 500 mM NaCl, and 0.1% NP-40, and 500 pmols of biotinylated histone peptides were loaded onto streptavidin coated magnetic beads before incubation with 40 pmols of protein. Bound proteins were detected with rabbit GST antibody (Sigma, G7781). Point mutants were generated by site-directed mutagenesis and purified/assayed as described above. The YEATS domains of Taf14, AF9, ENL, and GAS41 were previously described. Western blotting Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program. Dot blotting Increasing concentrations of biotinylated histone peptides (0.06–1.5 μg) were spotted onto a PVDF membrane then probed with the anti-H3K9ac (Millipore, 07-352) or H3K9cr (PTM Biolabs, PTM-516) at 1:2000 in a 5% non-fat milk solution and detected with an HRP-conjugated anti-rabbit by enhanced chemiluminesence (ECL). Bromodomains pull-downs cDNAs of GST-fused bromodomains were obtained either from EpiCypher Inc. or as a kind gift from Katrin Chua (Stanford University). GST fusions were expressed as described above except that the preparation buffer contained 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol (v/v), and 1 mM DTT. Pull-down assays were preformed as described above except that the assay buffer contained 50 mM Tris (pH 8.0), 300 mM NaCl, and 0.1% NP-40. Supplementary Material Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5IOK. Author contributions F.H.A., S.A.S., E.K.S., J.B.B., A.G., I.K.T and K.K. performed experiments and together with X.S., B.D.S and T.G.K. analyzed the data. F.H.A., S.A.S., B.D.S. and T.G.K. wrote the manuscript with input from all authors. Competing Financial Interest The authors declare no competing financial interests. Additional information Any supplementary information is available in the online version of this paper. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification Intracellular Crotonyl-CoA Stimulates Transcription through p300-Catalyzed Histone Crotonylation Protein lysine acylation and cysteine succination by intermediates of energy metabolism Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach Perceiving the epigenetic landscape through histone readers Interpreting the language of histone and DNA modifications A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications Histone recognition and large-scale structural analysis of the human bromodomain family YEATS domain proteins: a diverse family with many links to chromatin modification and transcription AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response Anc1 interacts with the catalytic subunits of the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes RSC and INO80, and the histone acetyltransferase complex NuA3 Preparation and analysis of the INO80 complex TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9 The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation Phaser crystallographic software PHENIX: a comprehensive Python-based system for macromolecular structure solution Features and development of Coot Molecular basis for chromatin binding and regulation of MLL5 Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4 A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery The structural mechanism for the recognition of H3K9cr (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine. H3K9cr is a selective target of the Taf14 YEATS domain (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. Total H3 was used as a loading control. (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. Spectra are color coded according to the protein:peptide molar ratio. (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides.","denotations":[{"span":{"begin":4,"end":9},"obj":"0.99879897,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"1"},{"span":{"begin":10,"end":22},"obj":"0.9920379,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"2"},{"span":{"begin":38,"end":45},"obj":"0.9768092,protein_type,cleaner0,2023-07-04T16:21:00Z,MESH:","id":"3"},{"span":{"begin":46,"end":59},"obj":"0.60347056,ptm,cleaner0,2023-07-04T16:18:43Z,MESH:","id":"4"},{"span":{"begin":81,"end":88},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"553"},{"span":{"begin":259,"end":271},"obj":"0.98157585,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"5"},{"span":{"begin":298,"end":305},"obj":"0.6092492,protein_type,cleaner0,2023-07-04T16:21:00Z,MESH:","id":"6"},{"span":{"begin":306,"end":312},"obj":"0.64678025,residue_name,cleaner0,2023-07-05T08:52:59Z,SO:","id":"7"},{"span":{"begin":313,"end":326},"obj":"0.7005873,ptm,cleaner0,2023-07-04T16:18:43Z,MESH:","id":"8"},{"span":{"begin":408,"end":413},"obj":"0.9987513,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"9"},{"span":{"begin":414,"end":426},"obj":"0.98611736,structure_element,cleaner0,2023-07-04T16:20:27Z,SO:","id":"10"},{"span":{"begin":435,"end":449},"obj":"0.99818015,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"11"},{"span":{"begin":503,"end":516},"obj":"0.8457508,structure_element,cleaner0,2023-07-05T08:49:39Z,SO:","id":"12"},{"span":{"begin":522,"end":536},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"542"},{"span":{"begin":560,"end":573},"obj":"0.97012514,ptm,cleaner0,2023-07-04T16:18:43Z,MESH:","id":"13"},{"span":{"begin":577,"end":583},"obj":"0.8647513,residue_name,cleaner0,2023-07-05T08:53:04Z,SO:","id":"14"},{"span":{"begin":594,"end":608},"obj":"0.99726224,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"15"},{"span":{"begin":610,"end":613},"obj":"0.9968893,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"16"},{"span":{"begin":653,"end":660},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"554"},{"span":{"begin":710,"end":719},"obj":"0.9958947,taxonomy_domain,cleaner0,2023-07-04T16:21:35Z,DUMMY:","id":"17"},{"span":{"begin":834,"end":848},"obj":"0.7653515,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"18"},{"span":{"begin":857,"end":864},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"555"},{"span":{"begin":865,"end":867},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:36:55Z","id":"666"},{"span":{"begin":867,"end":870},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:37:14Z","id":"667"},{"span":{"begin":886,"end":890},"obj":"0.98865753,protein,cleaner0,2023-07-04T16:20:32Z,PR:","id":"20"},{"span":{"begin":894,"end":919},"obj":"0.98868376,protein_type,cleaner0,2023-07-04T16:22:54Z,MESH:","id":"21"},{"span":{"begin":941,"end":952},"obj":"0.7823207,ptm,cleaner0,2023-07-04T16:18:49Z,MESH:","id":"22"},{"span":{"begin":1027,"end":1041},"obj":"0.9841765,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"23"},{"span":{"begin":1046,"end":1058},"obj":"0.9894874,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"24"},{"span":{"begin":1060,"end":1063},"obj":"0.98921156,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"25"},{"span":{"begin":1129,"end":1133},"obj":"0.9711801,protein,cleaner0,2023-07-04T16:20:33Z,PR:","id":"26"},{"span":{"begin":1144,"end":1151},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"557"},{"span":{"begin":1152,"end":1165},"obj":"0.9621592,ptm,cleaner0,2023-07-04T16:18:43Z,MESH:","id":"27"},{"span":{"begin":1258,"end":1262},"obj":"0.9124805,protein,cleaner0,2023-07-04T16:20:33Z,PR:","id":"28"},{"span":{"begin":1273,"end":1284},"obj":"0.87436926,ptm,cleaner0,2023-07-04T16:18:49Z,MESH:","id":"29"},{"span":{"begin":1339,"end":1353},"obj":"0.91712606,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"30"},{"span":{"begin":1358,"end":1370},"obj":"0.9561159,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"31"},{"span":{"begin":1453,"end":1465},"obj":"0.8240044,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"32"},{"span":{"begin":1539,"end":1553},"obj":"0.6335516,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"33"},{"span":{"begin":1609,"end":1621},"obj":"0.94788533,structure_element,cleaner0,2023-07-04T16:21:48Z,SO:","id":"34"},{"span":{"begin":1623,"end":1626},"obj":"0.8961469,structure_element,cleaner0,2023-07-04T16:21:53Z,SO:","id":"35"},{"span":{"begin":1655,"end":1657},"obj":"0.92061985,structure_element,cleaner0,2023-07-05T08:51:22Z,SO:","id":"36"},{"span":{"begin":1688,"end":1700},"obj":"0.9774638,protein_state,cleaner0,2023-07-04T16:22:06Z,DUMMY:","id":"37"},{"span":{"begin":1743,"end":1753},"obj":"0.98200285,protein_state,cleaner0,2023-07-04T16:22:11Z,DUMMY:","id":"39"},{"span":{"begin":1779,"end":1791},"obj":"0.7515075,structure_element,cleaner0,2023-07-04T16:21:48Z,SO:","id":"41"},{"span":{"begin":1832,"end":1846},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"543"},{"span":{"begin":1879,"end":1891},"obj":"0.9015483,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"42"},{"span":{"begin":1946,"end":1951},"obj":"0.9973253,structure_element,cleaner0,2023-07-05T08:51:28Z,SO:","id":"43"},{"span":{"begin":1953,"end":1957},"obj":"0.99829453,protein,cleaner0,2023-07-05T07:54:03Z,PR:","id":"44"},{"span":{"begin":1959,"end":1962},"obj":"0.99791545,protein,cleaner0,2023-07-05T07:54:08Z,PR:","id":"45"},{"span":{"begin":1964,"end":1967},"obj":"0.9983627,protein,cleaner0,2023-07-05T07:54:14Z,PR:","id":"46"},{"span":{"begin":1969,"end":1974},"obj":"0.9985991,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"47"},{"span":{"begin":1976,"end":1980},"obj":"0.9987198,protein,cleaner0,2023-07-05T07:54:25Z,PR:","id":"48"},{"span":{"begin":1993,"end":1998},"obj":"0.96409446,species,cleaner0,2023-07-04T16:22:25Z,MESH:","id":"49"},{"span":{"begin":1999,"end":2002},"obj":"0.99878556,protein,cleaner0,2023-07-05T07:54:15Z,PR:","id":"50"},{"span":{"begin":2007,"end":2012},"obj":"0.9893267,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"51"},{"span":{"begin":2013,"end":2018},"obj":"0.9988275,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"52"},{"span":{"begin":2050,"end":2057},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"558"},{"span":{"begin":2063,"end":2065},"obj":"protein_type,MESH:,cleaner0,2023-07-05T07:55:54Z","id":"585"},{"span":{"begin":2065,"end":2069},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T07:56:23Z","id":"589"},{"span":{"begin":2075,"end":2087},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"547"},{"span":{"begin":2112,"end":2115},"obj":"0.99856865,protein,cleaner0,2023-07-05T07:54:15Z,PR:","id":"54"},{"span":{"begin":2116,"end":2128},"obj":"0.9379061,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"55"},{"span":{"begin":2169,"end":2194},"obj":"0.9904277,protein_type,cleaner0,2023-07-04T16:22:40Z,MESH:","id":"56"},{"span":{"begin":2195,"end":2200},"obj":"0.99846435,protein,cleaner0,2023-07-05T07:57:04Z,PR:","id":"57"},{"span":{"begin":2204,"end":2206},"obj":"protein_type,MESH:,cleaner0,2023-07-05T07:56:42Z","id":"590"},{"span":{"begin":2206,"end":2210},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T07:56:57Z","id":"591"},{"span":{"begin":2240,"end":2245},"obj":"0.9973254,protein,cleaner0,2023-07-05T07:57:04Z,PR:","id":"59"},{"span":{"begin":2255,"end":2257},"obj":"protein_type,MESH:,cleaner0,2023-07-05T07:57:26Z","id":"592"},{"span":{"begin":2257,"end":2260},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T07:57:40Z","id":"593"},{"span":{"begin":2261,"end":2272},"obj":"ptm,MESH:,cleaner0,2023-07-05T07:57:58Z","id":"594"},{"span":{"begin":2360,"end":2365},"obj":"0.9963744,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"61"},{"span":{"begin":2378,"end":2390},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"548"},{"span":{"begin":2415,"end":2420},"obj":"0.9987602,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"62"},{"span":{"begin":2421,"end":2433},"obj":"0.94719505,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"63"},{"span":{"begin":2480,"end":2485},"obj":"0.9980909,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"64"},{"span":{"begin":2559,"end":2564},"obj":"0.84213173,complex_assembly,cleaner0,2023-07-04T16:23:51Z,GO:","id":"67"},{"span":{"begin":2569,"end":2574},"obj":"0.6936775,complex_assembly,cleaner0,2023-07-04T16:24:02Z,GO:","id":"68"},{"span":{"begin":2585,"end":2590},"obj":"0.9974746,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"69"},{"span":{"begin":2658,"end":2663},"obj":"0.7698535,complex_assembly,cleaner0,2023-07-05T07:58:48Z,GO:","id":"71"},{"span":{"begin":2665,"end":2672},"obj":"0.67576855,complex_assembly,cleaner0,2023-07-05T07:58:53Z,GO:","id":"72"},{"span":{"begin":2677,"end":2680},"obj":"0.5300177,complex_assembly,cleaner0,2023-07-05T07:59:00Z,GO:","id":"73"},{"span":{"begin":2690,"end":2715},"obj":"0.8560897,protein_type,cleaner0,2023-07-04T16:22:53Z,MESH:","id":"74"},{"span":{"begin":2724,"end":2728},"obj":"0.77899855,complex_assembly,cleaner0,2023-07-05T08:50:53Z,GO:","id":"76"},{"span":{"begin":2764,"end":2769},"obj":"0.99761933,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"77"},{"span":{"begin":2856,"end":2861},"obj":"0.9986816,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"78"},{"span":{"begin":2862,"end":2874},"obj":"0.93180734,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"79"},{"span":{"begin":2890,"end":2904},"obj":"0.9986914,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"80"},{"span":{"begin":2919,"end":2926},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"561"},{"span":{"begin":2927,"end":2929},"obj":"protein_type,MESH:,cleaner0,2023-07-05T07:55:54Z","id":"586"},{"span":{"begin":2930,"end":2942},"obj":"0.6754686,protein_state,cleaner0,2023-07-04T16:22:07Z,DUMMY:","id":"82"},{"span":{"begin":2946,"end":2954},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T07:58:27Z","id":"595"},{"span":{"begin":2956,"end":2958},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:00:12Z","id":"597"},{"span":{"begin":2958,"end":2962},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T07:59:41Z","id":"598"},{"span":{"begin":3015,"end":3017},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:00:12Z","id":"599"},{"span":{"begin":3017,"end":3021},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:00:29Z","id":"600"},{"span":{"begin":3036,"end":3041},"obj":"0.9966305,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"87"},{"span":{"begin":3128,"end":3130},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:00:50Z","id":"601"},{"span":{"begin":3130,"end":3134},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:01:02Z","id":"602"},{"span":{"begin":3155,"end":3172},"obj":"0.9973307,evidence,cleaner0,2023-07-05T08:56:20Z,DUMMY:","id":"89"},{"span":{"begin":3180,"end":3185},"obj":"0.9986278,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"90"},{"span":{"begin":3186,"end":3198},"obj":"0.9957862,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"91"},{"span":{"begin":3199,"end":3214},"obj":"0.9311703,protein_state,cleaner0,2023-07-05T08:53:17Z,DUMMY:","id":"92"},{"span":{"begin":3215,"end":3225},"obj":"0.97627616,chemical,cleaner0,2023-07-05T08:36:15Z,CHEBI:","id":"93"},{"span":{"begin":3236,"end":3240},"obj":"0.96639997,residue_range,cleaner0,2023-07-05T08:49:48Z,DUMMY:","id":"94"},{"span":{"begin":3244,"end":3246},"obj":"0.9895627,protein_type,cleaner0,2023-07-05T07:55:54Z,MESH:","id":"95"},{"span":{"begin":3341,"end":3346},"obj":"0.998461,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"97"},{"span":{"begin":3347,"end":3359},"obj":"0.9965588,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"98"},{"span":{"begin":3370,"end":3403},"obj":"0.99343634,structure_element,cleaner0,2023-07-05T08:51:32Z,SO:","id":"99"},{"span":{"begin":3421,"end":3444},"obj":"0.96191806,structure_element,cleaner0,2023-07-05T08:51:36Z,SO:","id":"100"},{"span":{"begin":3461,"end":3466},"obj":"0.6941337,structure_element,cleaner0,2023-07-05T08:51:44Z,SO:","id":"102"},{"span":{"begin":3479,"end":3491},"obj":"0.9979013,site,cleaner0,2023-07-05T08:51:58Z,SO:","id":"103"},{"span":{"begin":3496,"end":3498},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:01:22Z","id":"603"},{"span":{"begin":3498,"end":3502},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:01:34Z","id":"604"},{"span":{"begin":3518,"end":3520},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:01:47Z","id":"605"},{"span":{"begin":3520,"end":3524},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:01:58Z","id":"606"},{"span":{"begin":3544,"end":3565},"obj":"0.9527997,protein_state,cleaner0,2023-07-05T08:53:22Z,DUMMY:","id":"106"},{"span":{"begin":3602,"end":3611},"obj":"0.99544466,structure_element,cleaner0,2023-07-05T08:51:48Z,SO:","id":"107"},{"span":{"begin":3774,"end":3788},"obj":"0.9968951,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"108"},{"span":{"begin":3841,"end":3853},"obj":"0.884992,protein_state,cleaner0,2023-07-04T16:22:07Z,DUMMY:","id":"109"},{"span":{"begin":3854,"end":3860},"obj":"0.98747534,residue_name,cleaner0,2023-07-05T08:02:11Z,SO:","id":"110"},{"span":{"begin":3903,"end":3907},"obj":"0.99885833,residue_name_number,cleaner0,2023-07-05T08:02:22Z,DUMMY:","id":"112"},{"span":{"begin":3952,"end":3962},"obj":"0.9932809,structure_element,cleaner0,2023-07-05T08:52:02Z,SO:","id":"114"},{"span":{"begin":4090,"end":4095},"obj":"0.99896455,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"115"},{"span":{"begin":4100,"end":4105},"obj":"0.99897707,residue_name_number,cleaner0,2023-07-05T08:02:31Z,DUMMY:","id":"116"},{"span":{"begin":4465,"end":4470},"obj":"0.99895763,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"117"},{"span":{"begin":4709,"end":4714},"obj":"0.9987826,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"118"},{"span":{"begin":5333,"end":5338},"obj":"0.9991684,residue_name_number,cleaner0,2023-07-05T08:02:41Z,DUMMY:","id":"119"},{"span":{"begin":5368,"end":5373},"obj":"0.9991742,residue_name_number,cleaner0,2023-07-05T08:02:45Z,DUMMY:","id":"120"},{"span":{"begin":5411,"end":5416},"obj":"0.9991697,residue_name_number,cleaner0,2023-07-05T08:02:44Z,DUMMY:","id":"121"},{"span":{"begin":5485,"end":5489},"obj":"0.9990522,residue_name_number,cleaner0,2023-07-05T08:02:22Z,DUMMY:","id":"122"},{"span":{"begin":5538,"end":5543},"obj":"0.99915934,residue_name_number,cleaner0,2023-07-05T08:02:45Z,DUMMY:","id":"123"},{"span":{"begin":5621,"end":5625},"obj":"0.9990495,residue_name_number,cleaner0,2023-07-05T08:02:22Z,DUMMY:","id":"124"},{"span":{"begin":5686,"end":5691},"obj":"0.99916136,residue_name_number,cleaner0,2023-07-05T08:02:52Z,DUMMY:","id":"125"},{"span":{"begin":5716,"end":5720},"obj":"0.9988856,residue_name_number,cleaner0,2023-07-05T08:02:22Z,DUMMY:","id":"126"},{"span":{"begin":5816,"end":5821},"obj":"0.9991516,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"127"},{"span":{"begin":5836,"end":5841},"obj":"0.9917144,chemical,cleaner0,2023-07-05T08:55:01Z,CHEBI:","id":"128"},{"span":{"begin":5901,"end":5906},"obj":"0.99916196,residue_name_number,cleaner0,2023-07-05T08:02:57Z,DUMMY:","id":"129"},{"span":{"begin":5981,"end":5986},"obj":"0.9983511,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"130"},{"span":{"begin":5987,"end":6017},"obj":"0.9934926,site,cleaner0,2023-07-05T08:03:03Z,SO:","id":"131"},{"span":{"begin":6036,"end":6076},"obj":"0.9922565,experimental_method,cleaner0,2023-07-05T08:03:06Z,MESH:","id":"132"},{"span":{"begin":6149,"end":6154},"obj":"0.99884135,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"133"},{"span":{"begin":6155,"end":6167},"obj":"0.94870484,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"134"},{"span":{"begin":6171,"end":6173},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:03:24Z","id":"607"},{"span":{"begin":6173,"end":6177},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:03:37Z","id":"608"},{"span":{"begin":6193,"end":6214},"obj":"0.995806,evidence,cleaner0,2023-07-05T08:03:41Z,DUMMY:","id":"136"},{"span":{"begin":6216,"end":6218},"obj":"0.9937781,evidence,cleaner0,2023-07-05T08:03:45Z,DUMMY:","id":"137"},{"span":{"begin":6228,"end":6250},"obj":"0.9887676,complex_assembly,cleaner0,2023-07-05T08:35:55Z,GO:","id":"138"},{"span":{"begin":6298,"end":6323},"obj":"0.9937454,experimental_method,cleaner0,2023-07-05T08:04:07Z,MESH:","id":"139"},{"span":{"begin":6379,"end":6397},"obj":"0.99432206,evidence,cleaner0,2023-07-05T08:04:01Z,DUMMY:","id":"140"},{"span":{"begin":6497,"end":6499},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:04:40Z","id":"609"},{"span":{"begin":6499,"end":6503},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:04:52Z","id":"610"},{"span":{"begin":6519,"end":6524},"obj":"0.9988524,protein,cleaner0,2023-07-04T16:20:21Z,PR:","id":"143"},{"span":{"begin":6548,"end":6550},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:08:27Z","id":"611"},{"span":{"begin":6550,"end":6554},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:08:52Z","id":"612"},{"span":{"begin":6569,"end":6574},"obj":"0.99658173,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"145"},{"span":{"begin":6589,"end":6608},"obj":"0.8414014,experimental_method,cleaner0,2023-07-05T08:57:36Z,MESH:","id":"146"},{"span":{"begin":6638,"end":6643},"obj":"0.9963198,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"147"},{"span":{"begin":6672,"end":6693},"obj":"0.9293416,experimental_method,cleaner0,2023-07-05T08:57:39Z,MESH:","id":"148"},{"span":{"begin":6728,"end":6730},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:09:10Z","id":"613"},{"span":{"begin":6730,"end":6734},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:09:22Z","id":"614"},{"span":{"begin":6736,"end":6738},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:09:38Z","id":"615"},{"span":{"begin":6738,"end":6742},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:09:50Z","id":"616"},{"span":{"begin":6747,"end":6749},"obj":"0.7814144,protein_type,cleaner0,2023-07-05T07:55:54Z,MESH:","id":"151"},{"span":{"begin":6817,"end":6819},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:10:08Z","id":"617"},{"span":{"begin":6819,"end":6823},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:10:21Z","id":"618"},{"span":{"begin":6828,"end":6830},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:10:36Z","id":"619"},{"span":{"begin":6830,"end":6834},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:10:54Z","id":"620"},{"span":{"begin":6852,"end":6857},"obj":"0.99671257,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"154"},{"span":{"begin":6858,"end":6866},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:57:52Z","id":"690"},{"span":{"begin":6914,"end":6916},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:38:01Z","id":"668"},{"span":{"begin":6916,"end":6920},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:38:14Z","id":"669"},{"span":{"begin":6934,"end":6939},"obj":"0.9966935,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"156"},{"span":{"begin":6958,"end":6960},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:38:30Z","id":"670"},{"span":{"begin":6960,"end":6964},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:38:42Z","id":"671"},{"span":{"begin":6996,"end":7022},"obj":"0.99616396,protein_type,cleaner0,2023-07-05T08:06:46Z,MESH:","id":"158"},{"span":{"begin":7024,"end":7028},"obj":"0.9961572,protein_type,cleaner0,2023-07-05T08:06:50Z,MESH:","id":"159"},{"span":{"begin":7034,"end":7054},"obj":"0.99562824,protein_type,cleaner0,2023-07-05T08:07:02Z,MESH:","id":"160"},{"span":{"begin":7056,"end":7061},"obj":"0.9862266,protein_type,cleaner0,2023-07-05T08:07:06Z,MESH:","id":"161"},{"span":{"begin":7114,"end":7119},"obj":"0.9961592,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"163"},{"span":{"begin":7141,"end":7146},"obj":"0.99671113,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"164"},{"span":{"begin":7147,"end":7151},"obj":"0.9969458,protein_type,cleaner0,2023-07-05T08:06:51Z,MESH:","id":"165"},{"span":{"begin":7153,"end":7157},"obj":"0.99758315,protein,cleaner0,2023-07-05T08:07:12Z,PR:","id":"166"},{"span":{"begin":7159,"end":7163},"obj":"0.9978067,protein,cleaner0,2023-07-05T08:07:16Z,PR:","id":"167"},{"span":{"begin":7169,"end":7175},"obj":"0.9977192,protein,cleaner0,2023-07-05T08:07:20Z,PR:","id":"168"},{"span":{"begin":7180,"end":7185},"obj":"0.9930031,protein_type,cleaner0,2023-07-05T08:07:06Z,MESH:","id":"169"},{"span":{"begin":7187,"end":7191},"obj":"0.99792385,protein,cleaner0,2023-07-05T08:07:24Z,PR:","id":"170"},{"span":{"begin":7193,"end":7197},"obj":"0.99804014,protein,cleaner0,2023-07-05T08:07:27Z,PR:","id":"171"},{"span":{"begin":7203,"end":7207},"obj":"0.9978777,protein,cleaner0,2023-07-05T08:07:31Z,PR:","id":"172"},{"span":{"begin":7214,"end":7221},"obj":"0.53316224,experimental_method,cleaner0,2023-07-05T09:00:08Z,MESH:","id":"173"},{"span":{"begin":7275,"end":7277},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:11:11Z","id":"621"},{"span":{"begin":7277,"end":7281},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:11:24Z","id":"622"},{"span":{"begin":7335,"end":7338},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:58:28Z","id":"691"},{"span":{"begin":7339,"end":7347},"obj":"0.47926757,experimental_method,cleaner0,2023-07-05T09:00:12Z,MESH:","id":"175"},{"span":{"begin":7405,"end":7409},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:58:22Z","id":"692"},{"span":{"begin":7410,"end":7418},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T09:04:29Z","id":"695"},{"span":{"begin":7461,"end":7463},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:11:41Z","id":"623"},{"span":{"begin":7463,"end":7467},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:12:06Z","id":"624"},{"span":{"begin":7536,"end":7538},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:12:21Z","id":"625"},{"span":{"begin":7538,"end":7542},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:12:32Z","id":"626"},{"span":{"begin":7587,"end":7589},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:12:47Z","id":"627"},{"span":{"begin":7589,"end":7593},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:13:00Z","id":"628"},{"span":{"begin":7628,"end":7633},"obj":"0.9963509,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"179"},{"span":{"begin":7725,"end":7729},"obj":"0.9969043,protein_type,cleaner0,2023-07-05T08:06:51Z,MESH:","id":"180"},{"span":{"begin":7734,"end":7739},"obj":"0.9925954,protein_type,cleaner0,2023-07-05T08:07:06Z,MESH:","id":"181"},{"span":{"begin":7777,"end":7787},"obj":"protein_state,DUMMY:,cleaner0,2023-07-04T16:22:11Z","id":"584"},{"span":{"begin":7788,"end":7795},"obj":"protein_type,MESH:,cleaner0,2023-07-04T16:21:00Z","id":"565"},{"span":{"begin":7807,"end":7812},"obj":"0.9988348,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"182"},{"span":{"begin":7813,"end":7825},"obj":"0.9854789,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"183"},{"span":{"begin":7834,"end":7844},"obj":"0.9957682,protein_state,cleaner0,2023-07-04T16:22:11Z,DUMMY:","id":"184"},{"span":{"begin":7845,"end":7847},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:13:59Z","id":"630"},{"span":{"begin":7847,"end":7849},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:14:10Z","id":"631"},{"span":{"begin":7904,"end":7906},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:14:26Z","id":"632"},{"span":{"begin":7906,"end":7910},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:14:39Z","id":"633"},{"span":{"begin":7939,"end":7944},"obj":"0.99873716,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"187"},{"span":{"begin":7953,"end":7967},"obj":"0.9983891,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"188"},{"span":{"begin":7989,"end":8000},"obj":"0.9863785,experimental_method,cleaner0,2023-07-05T08:18:57Z,MESH:","id":"189"},{"span":{"begin":8030,"end":8040},"obj":"0.990856,chemical,cleaner0,2023-07-05T08:36:15Z,CHEBI:","id":"190"},{"span":{"begin":8044,"end":8054},"obj":"0.9934884,chemical,cleaner0,2023-07-05T08:38:55Z,CHEBI:","id":"191"},{"span":{"begin":8067,"end":8075},"obj":"0.96406466,experimental_method,cleaner0,2023-07-05T09:00:19Z,MESH:","id":"193"},{"span":{"begin":8085,"end":8096},"obj":"protein_state,DUMMY:,cleaner0,2023-07-05T08:53:55Z","id":"688"},{"span":{"begin":8097,"end":8102},"obj":"0.9987318,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"195"},{"span":{"begin":8103,"end":8115},"obj":"0.98387945,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"196"},{"span":{"begin":8167,"end":8169},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:15:14Z","id":"636"},{"span":{"begin":8169,"end":8173},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:15:26Z","id":"637"},{"span":{"begin":8182,"end":8199},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T08:59:29Z","id":"694"},{"span":{"begin":8231,"end":8234},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T09:04:29Z","id":"693"},{"span":{"begin":8305,"end":8307},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:15:40Z","id":"638"},{"span":{"begin":8307,"end":8311},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:15:54Z","id":"639"},{"span":{"begin":8412,"end":8422},"obj":"0.49922895,evidence,cleaner0,2023-07-05T08:56:27Z,DUMMY:","id":"199"},{"span":{"begin":8426,"end":8431},"obj":"0.99908006,residue_name_number,cleaner0,2023-07-05T08:15:58Z,DUMMY:","id":"200"},{"span":{"begin":8436,"end":8441},"obj":"0.9990513,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"201"},{"span":{"begin":8449,"end":8461},"obj":"0.98532,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"202"},{"span":{"begin":8489,"end":8491},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:16:15Z","id":"640"},{"span":{"begin":8491,"end":8495},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:16:28Z","id":"641"},{"span":{"begin":8500,"end":8502},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:16:48Z","id":"642"},{"span":{"begin":8502,"end":8506},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:16:59Z","id":"643"},{"span":{"begin":8570,"end":8617},"obj":"site,SO:,cleaner0,2023-07-05T08:13:40Z","id":"629"},{"span":{"begin":8684,"end":8689},"obj":"0.998936,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"207"},{"span":{"begin":8747,"end":8749},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:17:15Z","id":"644"},{"span":{"begin":8749,"end":8753},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:17:29Z","id":"645"},{"span":{"begin":8774,"end":8776},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:17:45Z","id":"646"},{"span":{"begin":8776,"end":8780},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:17:57Z","id":"647"},{"span":{"begin":8953,"end":8965},"obj":"0.99369735,complex_assembly,cleaner0,2023-07-05T08:18:01Z,GO:","id":"210"},{"span":{"begin":9001,"end":9006},"obj":"0.99862266,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"211"},{"span":{"begin":9007,"end":9019},"obj":"0.9849653,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"212"},{"span":{"begin":9067,"end":9077},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"696"},{"span":{"begin":9098,"end":9123},"obj":"0.99509495,experimental_method,cleaner0,2023-07-05T09:00:34Z,MESH:","id":"213"},{"span":{"begin":9130,"end":9132},"obj":"0.99270695,protein_type,cleaner0,2023-07-05T07:55:54Z,MESH:","id":"214"},{"span":{"begin":9142,"end":9152},"obj":"0.9932382,protein_state,cleaner0,2023-07-04T16:22:11Z,DUMMY:","id":"216"},{"span":{"begin":9154,"end":9167},"obj":"0.956428,protein_state,cleaner0,2023-07-05T08:40:11Z,DUMMY:","id":"217"},{"span":{"begin":9169,"end":9180},"obj":"0.9795297,protein_state,cleaner0,2023-07-05T08:40:15Z,DUMMY:","id":"218"},{"span":{"begin":9186,"end":9198},"obj":"0.97335297,protein_state,cleaner0,2023-07-04T16:22:07Z,DUMMY:","id":"219"},{"span":{"begin":9202,"end":9210},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:18:20Z","id":"648"},{"span":{"begin":9221,"end":9225},"obj":"0.9283707,residue_range,cleaner0,2023-07-05T08:50:16Z,DUMMY:","id":"222"},{"span":{"begin":9229,"end":9231},"obj":"0.951781,protein_type,cleaner0,2023-07-05T07:55:54Z,MESH:","id":"223"},{"span":{"begin":9287,"end":9292},"obj":"0.99854577,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"224"},{"span":{"begin":9293,"end":9305},"obj":"0.98509496,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"225"},{"span":{"begin":9329,"end":9339},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T08:33:09Z","id":"649"},{"span":{"begin":9362,"end":9370},"obj":"0.78338593,protein_state,cleaner0,2023-07-05T08:54:04Z,DUMMY:","id":"228"},{"span":{"begin":9408,"end":9410},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:20:37Z","id":"653"},{"span":{"begin":9410,"end":9414},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:20:48Z","id":"654"},{"span":{"begin":9420,"end":9422},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:21:11Z","id":"655"},{"span":{"begin":9422,"end":9426},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:21:22Z","id":"656"},{"span":{"begin":9428,"end":9430},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:21:37Z","id":"657"},{"span":{"begin":9430,"end":9434},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:21:50Z","id":"658"},{"span":{"begin":9439,"end":9441},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:22:10Z","id":"659"},{"span":{"begin":9441,"end":9445},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:22:27Z","id":"660"},{"span":{"begin":9463,"end":9496},"obj":"0.98849183,experimental_method,cleaner0,2023-07-05T08:19:00Z,MESH:","id":"234"},{"span":{"begin":9510,"end":9520},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T08:33:21Z","id":"650"},{"span":{"begin":9522,"end":9532},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T08:33:34Z","id":"651"},{"span":{"begin":9538,"end":9548},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T08:33:45Z","id":"652"},{"span":{"begin":9565,"end":9593},"obj":"evidence,DUMMY:,cleaner0,2023-07-05T09:01:01Z","id":"697"},{"span":{"begin":9601,"end":9606},"obj":"0.9984143,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"241"},{"span":{"begin":9607,"end":9619},"obj":"0.9821952,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"242"},{"span":{"begin":9734,"end":9744},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T08:33:09Z","id":"663"},{"span":{"begin":9796,"end":9798},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:22:49Z","id":"661"},{"span":{"begin":9798,"end":9802},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:23:04Z","id":"662"},{"span":{"begin":9848,"end":9879},"obj":"0.99294585,experimental_method,cleaner0,2023-07-05T09:01:12Z,MESH:","id":"246"},{"span":{"begin":9904,"end":9909},"obj":"0.9990103,residue_name_number,cleaner0,2023-07-05T08:15:59Z,DUMMY:","id":"248"},{"span":{"begin":9986,"end":10000},"obj":"0.9981839,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"249"},{"span":{"begin":10145,"end":10152},"obj":"0.99031365,protein_state,cleaner0,2023-07-05T08:49:33Z,DUMMY:","id":"252"},{"span":{"begin":10153,"end":10158},"obj":"0.9989976,residue_name_number,cleaner0,2023-07-05T08:15:59Z,DUMMY:","id":"253"},{"span":{"begin":10195,"end":10205},"obj":"protein_state,DUMMY:,cleaner0,2023-07-05T09:01:51Z","id":"698"},{"span":{"begin":10206,"end":10211},"obj":"0.9989625,residue_name_number,cleaner0,2023-07-05T08:15:59Z,DUMMY:","id":"254"},{"span":{"begin":10257,"end":10265},"obj":"0.885494,protein_state,cleaner0,2023-07-05T08:54:04Z,DUMMY:","id":"255"},{"span":{"begin":10291,"end":10296},"obj":"0.9990196,residue_name_number,cleaner0,2023-07-05T08:15:59Z,DUMMY:","id":"257"},{"span":{"begin":10354,"end":10362},"obj":"0.9797934,experimental_method,cleaner0,2023-07-05T09:01:59Z,MESH:","id":"258"},{"span":{"begin":10366,"end":10371},"obj":"0.99903935,residue_name_number,cleaner0,2023-07-05T08:24:25Z,DUMMY:","id":"259"},{"span":{"begin":10410,"end":10415},"obj":"0.9990657,residue_name_number,cleaner0,2023-07-05T08:02:27Z,DUMMY:","id":"260"},{"span":{"begin":10515,"end":10529},"obj":"0.99843556,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"261"},{"span":{"begin":10533,"end":10542},"obj":"0.984743,protein_state,cleaner0,2023-07-05T08:54:12Z,DUMMY:","id":"262"},{"span":{"begin":10554,"end":10559},"obj":"0.99253696,species,cleaner0,2023-07-04T16:22:25Z,MESH:","id":"263"},{"span":{"begin":10560,"end":10573},"obj":"structure_element,SO:,cleaner0,2023-07-05T08:49:39Z","id":"684"},{"span":{"begin":10577,"end":10598},"obj":"0.9765474,experimental_method,cleaner0,2023-07-05T09:02:03Z,MESH:","id":"265"},{"span":{"begin":10625,"end":10635},"obj":"0.9461614,protein_state,cleaner0,2023-07-04T16:22:11Z,DUMMY:","id":"267"},{"span":{"begin":10637,"end":10650},"obj":"0.9879154,protein_state,cleaner0,2023-07-05T08:40:11Z,DUMMY:","id":"268"},{"span":{"begin":10652,"end":10663},"obj":"0.9869178,protein_state,cleaner0,2023-07-05T08:40:15Z,DUMMY:","id":"269"},{"span":{"begin":10669,"end":10681},"obj":"0.9762997,protein_state,cleaner0,2023-07-04T16:22:07Z,DUMMY:","id":"270"},{"span":{"begin":10682,"end":10689},"obj":"protein_type,MESH:,cleaner0,2023-07-05T09:02:17Z","id":"699"},{"span":{"begin":10741,"end":10754},"obj":"structure_element,SO:,cleaner0,2023-07-05T08:49:39Z","id":"685"},{"span":{"begin":10788,"end":10802},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"544"},{"span":{"begin":10883,"end":10889},"obj":"0.9987778,protein,cleaner0,2023-07-05T08:39:48Z,PR:","id":"275"},{"span":{"begin":10894,"end":10897},"obj":"0.99860966,protein,cleaner0,2023-07-05T07:54:09Z,PR:","id":"276"},{"span":{"begin":10925,"end":10937},"obj":"0.9837325,protein_state,cleaner0,2023-07-04T16:22:07Z,DUMMY:","id":"277"},{"span":{"begin":10948,"end":10953},"obj":"0.99857676,protein,cleaner0,2023-07-05T08:39:56Z,PR:","id":"279"},{"span":{"begin":10958,"end":10961},"obj":"0.9983223,protein,cleaner0,2023-07-05T07:54:15Z,PR:","id":"280"},{"span":{"begin":10968,"end":10976},"obj":"0.99105185,protein_state,cleaner0,2023-07-05T08:54:04Z,DUMMY:","id":"281"},{"span":{"begin":11018,"end":11030},"obj":"0.9855747,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"283"},{"span":{"begin":11040,"end":11059},"obj":"0.80789137,protein_type,cleaner0,2023-07-05T08:52:52Z,MESH:","id":"284"},{"span":{"begin":11061,"end":11072},"obj":"0.9922646,structure_element,cleaner0,2023-07-05T08:52:07Z,SO:","id":"285"},{"span":{"begin":11093,"end":11107},"obj":"0.99855334,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"286"},{"span":{"begin":11135,"end":11138},"obj":"0.7588632,structure_element,cleaner0,2023-07-04T16:21:53Z,SO:","id":"287"},{"span":{"begin":11142,"end":11163},"obj":"0.9541701,experimental_method,cleaner0,2023-07-05T09:02:34Z,MESH:","id":"288"},{"span":{"begin":11214,"end":11226},"obj":"0.99803704,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"289"},{"span":{"begin":11231,"end":11246},"obj":"0.9968952,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"290"},{"span":{"begin":11300,"end":11312},"obj":"0.9787652,structure_element,cleaner0,2023-07-04T16:21:48Z,SO:","id":"291"},{"span":{"begin":11400,"end":11414},"obj":"0.99844176,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"292"},{"span":{"begin":11434,"end":11437},"obj":"0.8739639,structure_element,cleaner0,2023-07-04T16:21:53Z,SO:","id":"293"},{"span":{"begin":11441,"end":11445},"obj":"0.99858606,protein,cleaner0,2023-07-05T08:51:13Z,PR:","id":"294"},{"span":{"begin":11450,"end":11454},"obj":"0.9986286,protein,cleaner0,2023-07-05T08:51:16Z,PR:","id":"295"},{"span":{"begin":11518,"end":11530},"obj":"0.98622835,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"296"},{"span":{"begin":11563,"end":11577},"obj":"0.9985879,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"297"},{"span":{"begin":11617,"end":11629},"obj":"0.99071395,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"298"},{"span":{"begin":11633,"end":11638},"obj":"0.9987459,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"299"},{"span":{"begin":11662,"end":11669},"obj":"0.5421235,protein_type,cleaner0,2023-07-04T16:21:00Z,MESH:","id":"300"},{"span":{"begin":11670,"end":11683},"obj":"0.672082,ptm,cleaner0,2023-07-04T16:18:43Z,MESH:","id":"301"},{"span":{"begin":11870,"end":11872},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:41:07Z","id":"672"},{"span":{"begin":11872,"end":11876},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:41:21Z","id":"673"},{"span":{"begin":11887,"end":11892},"obj":"0.99646616,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"303"},{"span":{"begin":11925,"end":11929},"obj":"0.9973616,protein_type,cleaner0,2023-07-05T08:06:51Z,MESH:","id":"304"},{"span":{"begin":11934,"end":11939},"obj":"0.9948607,protein_type,cleaner0,2023-07-05T08:07:06Z,MESH:","id":"305"},{"span":{"begin":11983,"end":11993},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"700"},{"span":{"begin":12009,"end":12014},"obj":"0.998776,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"306"},{"span":{"begin":12015,"end":12027},"obj":"0.9936651,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"307"},{"span":{"begin":12151,"end":12165},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"545"},{"span":{"begin":12217,"end":12222},"obj":"0.9987233,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"308"},{"span":{"begin":12250,"end":12264},"obj":"0.99826485,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"309"},{"span":{"begin":12269,"end":12281},"obj":"0.9959139,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"310"},{"span":{"begin":12341,"end":12355},"obj":"residue_name,SO:,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z","id":"546"},{"span":{"begin":12377,"end":12382},"obj":"0.9981281,protein_type,cleaner0,2023-07-05T08:52:37Z,MESH:","id":"311"},{"span":{"begin":20558,"end":20560},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:46:18Z","id":"674"},{"span":{"begin":20560,"end":20564},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:46:30Z","id":"675"},{"span":{"begin":20591,"end":20605},"obj":"0.99838877,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"502"},{"span":{"begin":20615,"end":20632},"obj":"0.99736893,evidence,cleaner0,2023-07-05T08:56:31Z,DUMMY:","id":"503"},{"span":{"begin":20640,"end":20645},"obj":"0.99835616,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"504"},{"span":{"begin":20646,"end":20658},"obj":"0.97807664,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"505"},{"span":{"begin":20667,"end":20682},"obj":"protein_state,DUMMY:,cleaner0,2023-07-05T08:54:40Z","id":"689"},{"span":{"begin":20687,"end":20697},"obj":"0.80304146,chemical,cleaner0,2023-07-05T08:36:15Z,CHEBI:","id":"507"},{"span":{"begin":20719,"end":20721},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:46:58Z","id":"676"},{"span":{"begin":20721,"end":20725},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:47:12Z","id":"677"},{"span":{"begin":20829,"end":20834},"obj":"0.99865216,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"510"},{"span":{"begin":20835,"end":20847},"obj":"0.9790957,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"511"},{"span":{"begin":20913,"end":20927},"obj":"0.9983652,residue_name,melaniev@ebi.ac.uk,2023-07-06T15:25:20Z,SO:","id":"512"},{"span":{"begin":20932,"end":20934},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:47:40Z","id":"678"},{"span":{"begin":20934,"end":20938},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:47:55Z","id":"679"},{"span":{"begin":20968,"end":20973},"obj":"0.9988475,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"514"},{"span":{"begin":20974,"end":20986},"obj":"0.9892417,structure_element,cleaner0,2023-07-04T16:20:28Z,SO:","id":"515"},{"span":{"begin":20994,"end":21006},"obj":"0.98307806,experimental_method,cleaner0,2023-07-05T09:03:32Z,MESH:","id":"516"},{"span":{"begin":21040,"end":21042},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:48:14Z","id":"680"},{"span":{"begin":21042,"end":21046},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:48:26Z","id":"681"},{"span":{"begin":21051,"end":21053},"obj":"protein_type,MESH:,cleaner0,2023-07-05T08:48:42Z","id":"682"},{"span":{"begin":21053,"end":21057},"obj":"residue_name_number,DUMMY:,cleaner0,2023-07-05T08:48:52Z","id":"683"},{"span":{"begin":21061,"end":21070},"obj":"0.9924296,protein_state,cleaner0,2023-07-05T08:49:13Z,DUMMY:","id":"519"},{"span":{"begin":21072,"end":21074},"obj":"0.98962665,protein_state,cleaner0,2023-07-05T08:49:18Z,DUMMY:","id":"520"},{"span":{"begin":21094,"end":21098},"obj":"protein_type,MESH:,cleaner0,2023-07-05T09:03:46Z","id":"701"},{"span":{"begin":21099,"end":21107},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T09:04:00Z","id":"702"},{"span":{"begin":21108,"end":21113},"obj":"0.99134904,taxonomy_domain,cleaner0,2023-07-04T16:22:20Z,DUMMY:","id":"521"},{"span":{"begin":21129,"end":21131},"obj":"0.9896226,protein_type,cleaner0,2023-07-05T07:55:54Z,MESH:","id":"522"},{"span":{"begin":21180,"end":21191},"obj":"0.90694416,experimental_method,cleaner0,2023-07-05T09:03:35Z,MESH:","id":"523"},{"span":{"begin":21192,"end":21199},"obj":"0.65750146,evidence,cleaner0,2023-07-05T08:57:05Z,DUMMY:","id":"524"},{"span":{"begin":21203,"end":21208},"obj":"0.9981918,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"525"},{"span":{"begin":21209,"end":21214},"obj":"0.99271107,structure_element,cleaner0,2023-07-05T08:52:13Z,SO:","id":"526"},{"span":{"begin":21227,"end":21237},"obj":"0.7894704,chemical,cleaner0,2023-07-05T08:36:15Z,CHEBI:","id":"527"},{"span":{"begin":21242,"end":21252},"obj":"chemical,CHEBI:,cleaner0,2023-07-05T08:38:55Z","id":"635"},{"span":{"begin":21267,"end":21275},"obj":"experimental_method,MESH:,cleaner0,2023-07-05T09:04:10Z","id":"703"},{"span":{"begin":21280,"end":21287},"obj":"0.6348235,evidence,cleaner0,2023-07-05T08:57:07Z,DUMMY:","id":"531"},{"span":{"begin":21354,"end":21366},"obj":"0.98869205,experimental_method,cleaner0,2023-07-05T08:49:22Z,MESH:","id":"532"},{"span":{"begin":21379,"end":21403},"obj":"0.99291915,experimental_method,cleaner0,2023-07-05T08:49:24Z,MESH:","id":"533"},{"span":{"begin":21410,"end":21419},"obj":"0.99699277,protein_state,cleaner0,2023-07-05T08:49:30Z,DUMMY:","id":"534"},{"span":{"begin":21424,"end":21431},"obj":"0.959159,protein_state,cleaner0,2023-07-05T08:49:33Z,DUMMY:","id":"535"},{"span":{"begin":21432,"end":21437},"obj":"0.9981369,protein,cleaner0,2023-07-04T16:20:22Z,PR:","id":"536"},{"span":{"begin":21438,"end":21451},"obj":"0.981699,structure_element,cleaner0,2023-07-05T08:49:39Z,SO:","id":"537"}],"relations":[]}]

raw_BioC_XML/PMC4772114_raw.xml

@@ -0,0 +1,4 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>4772114</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep22324</infon><infon key="article-id_pii">srep22324</infon><infon key="article-id_pmc">4772114</infon><infon key="article-id_pmid">26927947</infon><infon key="elocation-id">22324</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Yokogawa;given-names:Mariko</infon><infon key="name_1">surname:Tsushima;given-names:Takashi</infon><infon key="name_2">surname:Noda;given-names:Nobuo N.</infon><infon key="name_3">surname:Kumeta;given-names:Hiroyuki</infon><infon key="name_4">surname:Enokizono;given-names:Yoshiaki</infon><infon key="name_5">surname:Yamashita;given-names:Kazuo</infon><infon key="name_6">surname:Standley;given-names:Daron M.</infon><infon key="name_7">surname:Takeuchi;given-names:Osamu</infon><infon key="name_8">surname:Akira;given-names:Shizuo</infon><infon key="name_9">surname:Inagaki;given-names:Fuyuhiko</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>101</offset><text>Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1084</offset><text>The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. Since aberrant activation of this system leads to auto immune disorders, it must be tightly regulated. Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1904</offset><text>Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2729</offset><text>Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>3441</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>3449</offset><text>Domain structures of Regnase-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>3480</offset><text>We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. This suggests that the PIN and ZF domains exist independently without interacting with each other. The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4303</offset><text>Contribution of each domain of Regnase-1 to the mRNA binding activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4373</offset><text>Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5661</offset><text>Contribution of each domain of Regnase-1 to RNase activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5720</offset><text>In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. The apparent half-life (T1/2) of the RNase activity was about 20 minutes. Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 &gt; 90 minutes). It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6810</offset><text>Dimer formation of the PIN domains</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6845</offset><text>During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8692</offset><text>Domain-domain interaction between the NTD and the PIN domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8753</offset><text>While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). These results clearly indicate a direct interaction between the PIN domain and the NTD. Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10692</offset><text>Residues critical for Regnase-1 RNase activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10739</offset><text>To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12527</offset><text>Molecular mechanism of target mRNA cleavage by the PIN dimer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12588</offset><text>Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). These were paired with a DDNN mutant that had no RNase activity by itself. When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14859</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14870</offset><text>We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15945</offset><text>The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>17024</offset><text>Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>17888</offset><text>The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19145</offset><text>Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19757</offset><text>The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>20555</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20563</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20599</offset><text>The DNA fragment encoding Regnase-1 derived from Mus musculus was cloned into pGEX6p vector (GE Healthcare). All the mutants were generated by PCR-mediated site-directed mutagenesis and confirmed by the DNA sequence analyses. As a catalytically deficient mutant, both Asp226 and Asp244 at the catalytic center of PIN were mutated to Asn, which is referred to as DDNN mutant. Regnase-1 was expressed at 16 °C using the Escherichia coli RosettaTM(DE3)pLysS strain. After purification with a GST-affinity resin, an N-terminal GST tag was digested by HRV-3 C protease. NTD was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). The other domains were further purified by cation exchange chromatography using Resource S (GE Healthcare), followed by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). Uniformly 15N or 13C, 15N-double labeled proteins for NMR experiments were prepared by growing E. coli host in M9 minimal medium containing 15NH4Cl, unlabeled glucose and 15N CELTONE® Base Powder (CIL) or 15NH4Cl, 13C6-glucose, and13C, 15N CELTONE® Base Powder (CIL), respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21771</offset><text>X-ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21793</offset><text>Crystallization was performed using the sitting drop vapor diffusion method at 20 °C and two crystal forms (I and II) were obtained. In the case of form I crystals, drops (0.5 μl) of 6 mg/ml selenomethionine-labeled Regnase-1 PIN-ZF (residues 134–339 derived from Mus musculus) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1 M (NH4)2HPO4, 200 mM NaCl and 100 mM sodium citrate (pH 5.5) whereas in the case of form II crystals, drops (0.5 μl) of 6 mg/ml native Regnase-1 PIN-ZF (residues 134–339) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1.7 M NaCl and 100 mM HEPES-NaOH (pH 7.0). Diffraction data were collected at a Photon Factory Advanced Ring beamline NE3A (form I) or at a SPring-8 beamline BL41XU (form II), and were processed with HKL2000. The structure of the form I crystal was determined by the multiple anomalous dispersion (MAD) method. Nine Se sites were found using the program SOLVE; however, the electron density obtained by MAD phases calculated using SOLVE was not good enough to build a model even after density modification using the program RESOLVE. Then the program CNS was used to find additional three Se sites and calculate MAD phases using 12 Se sites. The electron density after density modification using CNS was good enough to build a model. Structure of the form II crystal was determined by the molecular replacement method using CNS and using the structure of the form I crystal as a search model. For all structures, further model building was performed manually with COOT, and TLS and restrained refinement using isotropic individual B factors was performed with REFMAC5 in the CCP4 program suite. Crystallographic parameters are summarized in Supplementary Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23654</offset><text>NMR measurements</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23671</offset><text>All NMR experiments were carried out at 298 K on Inova 500-MHz, 600-MHz, and 800-MHz spectrometer (Agilent). The NMR data were processed using the NMRPipe, the Olivia (fermi.pharm.hokudai.ac.jp/olivia/), and the Sparky program (Sparky3, University of California, San Francisco).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23952</offset><text>For structure calculation, NOE distance restraints were obtained from 3D 15N-NOESY-HSQC (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD) and 13C-NOESY-HSQC spectra (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD). The NMR structures were determined using the CANDID/CYANA2.1. Dihedral restraints were derived from backbone chemical shifts using TALOS.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24378</offset><text>For the domain-domain interaction analyses between the NTD and the PIN domain, 1H-15N HSQC spectra of uniformly 15N-labeled proteins in the concentration of 100 μM were obtained in the presence of 3 or 6 molar equivalents of unlabeled proteins.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24626</offset><text>Preparation of RNAs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24646</offset><text>The fluorescently labeled RNAs at the 5′-end by 6-FAM were purchased from SIGMA-ALDORICH. The RNA sequences used in this study were shown below.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24793</offset><text>IL-6 mRNA 3′UTR (82–106): 5′-UGUUGUUCUCUACGAAGAACUGACA-3′ (25 nts)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24868</offset><text>Regnase-1 mRNA 3′UTR (191–211): 5′- CUGUUGAUACACAUUGUAUCU-3′ (21 nts)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24946</offset><text>Electrophoretic mobility shift assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24983</offset><text>Catalytically deficient Regnase-1 proteins, containing DDNN mutations, and 5′-terminally 6-FAM labeled RNAs were incubated in the RNA-binding buffer (20 mM HEPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM DTT, 10% glycerol (v/v), and 0.1% NP-40 (v/v)) at 4 °C for 30 minutes, then analyzed by non-denaturing polyacrylamide gel electrophoresis. The electrophoreses were performed at 4 °C using the 7.5% polyacrylamide (w/v) gel (monomer : bis = 29 : 1) in the electrophoresis buffer (25 mM Tris-HCl (pH 7.5) and 200 mM glycine). The fluorescence of 6-FAM labeled RNA was directly detected at the excitation wavelength of 460 nm with a fluorescence filter (Y515-Di) using a fluoroimaging analyzer (LAS-4000 (FUJIFILM)). The fluorescence intensity of each sample was quantified using ImageJ software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25797</offset><text>In vitro RNA cleavage assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25825</offset><text>Regnase-1 (2 μM) and 5′-terminally 6-FAM labeled RNA (1 μM) were incubated in the RNA-cleavage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT) at 37 °C. For the assay using combinations of Regnase-1 mutants, equimolar amounts of Regnase-1 mutants (2 μM each) were mixed with fluorescently labeled RNA (1 μM). After incubation for 30–120 minutes, the reaction was stopped by the addition of 1.5-fold volume of denaturing buffer containing 8 M urea and 100 mM EDTA, and samples were boiled. The electrophoreses were performed at room temperature using the 8 M urea containing denaturing gel with 20% polyacrylamide (w/v) (monomer : bis = 19 : 1) in 0.5 × TBE as the electrophoresis buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26581</offset><text>Docking calculations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26602</offset><text>For docking NTD to PIN, OSCAR-star was first used to rebuild sidechains in the head-to-tail PIN dimer. Docking was carried out by surFit (http://sysimm.ifrec.osaka-u.ac.jp/docking/main/) with restraints obtained from NMR data (Fig. 3a,b) as follows. NTD: R56, L58, G59, V86, K87, H88; PIN: V177, F210, T211, R214, F228, I229, V230, K231, L232, F234, D235, S236. Top-scoring model was selected.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26996</offset><text>For docking IL-6 mRNA 3′UTR to the PIN dimer, each domain of the PIN dimer structure was superimposed onto the PIN dimer of the human X-ray structure (PDB ID: 3V34) in order to graft both water molecules and Mg2+ ions to the mouse model. Each IL-6 representative structure was submitted to the HADDOCK 2.0 server, for total of 10 independent jobs. In order to be consistent with the cleavage assay, active residues consisted of all nucleotides in RNA, Mg2+ and W182, R183, K184, R188, R214, R215, K219, R220, and R247 in the protein. Docked models were selected based on the following criteria: one heavy atom within 7, 8, or 9th nucleotide from the 5′ end was &lt;5 Å from the Mg2+ ion on the primary PIN. Further classification was done manually in order to divide the selected models into two clusters.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27806</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27829</offset><text>Accession codes: The crystal structure of the Regnase-1 PIN domain has been deposited in the Protein Data Bank (accession codes: 5H9V (Form I) and 5H9W (Form II)). The chemical shift assignments of the NTD, the ZF domain, and the CTD have been deposited at Biological Magnetic Resonance Bank (accession codes: 25718, 25719, and 25720, respectively), and the coordinates for the ensemble have been deposited in the Protein Data Bank (accession codes: 2N5J, 2N5K, and 2N5L, respectively). </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28317</offset><text>How to cite this article: Yokogawa, M. et al. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci. Rep. 6, 22324; doi: 10.1038/srep22324 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>28516</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">783</infon><infon key="lpage">801</infon><infon key="name_0">surname:Akira;given-names:S.</infon><infon key="name_1">surname:Uematsu;given-names:S.</infon><infon key="name_2">surname:Takeuchi;given-names:O.</infon><infon key="pub-id_pmid">16497588</infon><infon key="section_type">REF</infon><infon key="source">Cell,</infon><infon key="type">ref</infon><infon key="volume">124</infon><infon key="year">2006</infon><offset>28539</offset><text>Pathogen recognition and innate immunity.</text></passage><passage><infon key="fpage">819</infon><infon key="lpage">26</infon><infon key="name_0">surname:Medzhitov;given-names:R.</infon><infon key="pub-id_pmid">17943118</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">449</infon><infon key="year">2007</infon><offset>28581</offset><text>Recognition of microorganisms and activation of the immune response</text></passage><passage><infon key="fpage">353</infon><infon key="lpage">89</infon><infon key="name_0">surname:Beutler;given-names:B.</infon><infon key="pub-id_pmid">16551253</infon><infon key="section_type">REF</infon><infon key="source">Annu Rev Immunol</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2006</infon><offset>28649</offset><text>Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large</text></passage><passage><infon key="fpage">1185</infon><infon key="lpage">90</infon><infon key="name_0">surname:Matsushita;given-names:K.</infon><infon key="pub-id_pmid">19322177</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">458</infon><infon key="year">2009</infon><offset>28737</offset><text>Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay</text></passage><passage><infon key="fpage">7386</infon><infon key="lpage">99</infon><infon key="name_0">surname:Mizgalska;given-names:D.</infon><infon key="pub-id_pmid">19909337</infon><infon key="section_type">REF</infon><infon key="source">FEBS J</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">2009</infon><offset>28825</offset><text>Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA</text></passage><passage><infon key="fpage">e49841</infon><infon key="name_0">surname:Li;given-names:M.</infon><infon key="pub-id_pmid">23185455</infon><infon key="section_type">REF</infon><infon key="source">PLoS One,</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2012</infon><offset>28962</offset><text>MCPIP1 down-regulates IL-2 expression through an ARE-independent pathway</text></passage><passage><infon key="fpage">708</infon><infon key="lpage">13</infon><infon key="name_0">surname:Uehata;given-names:T.</infon><infon key="name_1">surname:Akira;given-names:S.</infon><infon key="pub-id_pmid">23500036</infon><infon key="section_type">REF</infon><infon key="source">Biochim Biophys Acta</infon><infon key="type">ref</infon><infon key="volume">1829</infon><infon key="year">2013</infon><offset>29035</offset><text>mRNA degradation by the endoribonuclease Regnase-1/ZC3H12a/MCPIP-1</text></passage><passage><infon key="fpage">1167</infon><infon key="lpage">75</infon><infon key="name_0">surname:Iwasaki;given-names:H.</infon><infon key="pub-id_pmid">22037600</infon><infon key="section_type">REF</infon><infon key="source">Nat Immunol</infon><infon key="type">ref</infon><infon key="volume">12</infon><infon key="year">2011</infon><offset>29102</offset><text>The IκB kinase complex regulates the stability of cytokine-encoding mRNA induced by TLR-IL-1R by controlling degradation of regnase-1</text></passage><passage><infon key="fpage">6337</infon><infon key="lpage">46</infon><infon key="name_0">surname:Liang;given-names:J.</infon><infon key="pub-id_pmid">18178554</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>29240</offset><text>A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages</text></passage><passage><infon key="fpage">903</infon><infon key="lpage">10</infon><infon key="name_0">surname:Xu;given-names:J.</infon><infon key="name_1">surname:Fu;given-names:S.</infon><infon key="name_2">surname:Peng;given-names:W.</infon><infon key="name_3">surname:Rao;given-names:Z.</infon><infon key="pub-id_pmid">23132255</infon><infon key="section_type">REF</infon><infon key="source">Protein Cell</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2012</infon><offset>29332</offset><text>MCP-1-induced protein-1, an immune regulator</text></passage><passage><infon key="fpage">6957</infon><infon key="lpage">65</infon><infon key="name_0">surname:Xu;given-names:J.</infon><infon key="pub-id_pmid">22561375</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2012</infon><offset>29377</offset><text>Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase</text></passage><passage><infon key="fpage">685</infon><infon key="lpage">93</infon><infon key="name_0">surname:Hake;given-names:L. E.</infon><infon key="name_1">surname:Mendez;given-names:R.</infon><infon key="name_2">surname:Richter;given-names:J. D.</infon><infon key="pub-id_pmid">9447964</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell Biol</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1998</infon><offset>29457</offset><text>Specificity of RNA binding by CPEB: requirement for RNA recognition motifs and a novel zinc finger</text></passage><passage><infon key="fpage">9606</infon><infon key="lpage">13</infon><infon key="name_0">surname:Lai;given-names:W. S.</infon><infon key="name_1">surname:Kennington;given-names:E. A.</infon><infon key="name_2">surname:Blackshear;given-names:P. J.</infon><infon key="pub-id_pmid">11782475</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">2002</infon><offset>29556</offset><text>Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs</text></passage><passage><infon key="fpage">257</infon><infon key="lpage">64</infon><infon key="name_0">surname:Hudson;given-names:B. P.</infon><infon key="name_1">surname:Martinez-Yamout;given-names:M. A.</infon><infon key="name_2">surname:Dyson;given-names:H. J.</infon><infon key="name_3">surname:Wright;given-names:P. E.</infon><infon key="pub-id_pmid">14981510</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2004</infon><offset>29723</offset><text>Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d</text></passage><passage><infon key="fpage">367</infon><infon key="lpage">73</infon><infon key="name_0">surname:Hall;given-names:T. M.</infon><infon key="pub-id_pmid">15963892</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Struct Biol</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2005</infon><offset>29799</offset><text>Multiple modes of RNA recognition by zinc finger proteins</text></passage><passage><infon key="fpage">1177</infon><infon key="lpage">85</infon><infon key="name_0">surname:Zhou;given-names:L.</infon><infon key="pub-id_pmid">16574901</infon><infon key="section_type">REF</infon><infon key="source">Circ Res</infon><infon key="type">ref</infon><infon key="volume">98</infon><infon key="year">2006</infon><offset>29857</offset><text>Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction</text></passage><passage><infon key="fpage">2959</infon><infon key="lpage">73</infon><infon key="name_0">surname:Liang;given-names:J.</infon><infon key="pub-id_pmid">21115689</infon><infon key="section_type">REF</infon><infon key="source">J Exp Med</infon><infon key="type">ref</infon><infon key="volume">207</infon><infon key="year">2010</infon><offset>29995</offset><text>MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-kappaB signaling</text></passage><passage><infon key="fpage">W545</infon><infon key="lpage">9</infon><infon key="name_0">surname:Holm;given-names:L.</infon><infon key="name_1">surname:Rosenström;given-names:P.</infon><infon key="pub-id_pmid">20457744</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">2010</infon><offset>30100</offset><text>Dali server: conservation mapping in 3D</text></passage><passage><infon key="fpage">1036</infon><infon key="lpage">49</infon><infon key="name_0">surname:Uehata;given-names:T.</infon><infon key="pub-id_pmid">23706741</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">153</infon><infon key="year">2013</infon><offset>30140</offset><text>Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation</text></passage><passage><infon key="fpage">307</infon><infon key="lpage">326</infon><infon key="name_0">surname:Otwinowski;given-names:Z.</infon><infon key="name_1">surname:Minor;given-names:W.</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">1997</infon><offset>30229</offset><text>Processing of X-ray Diffraction Data Collected in Oscillation Mode</text></passage><passage><infon key="fpage">849</infon><infon key="lpage">61</infon><infon key="name_0">surname:Terwilliger;given-names:T. C.</infon><infon key="name_1">surname:Berendzen;given-names:J.</infon><infon key="pub-id_pmid">10089316</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">1999</infon><offset>30296</offset><text>Automated MAD and MIR structure solution</text></passage><passage><infon key="fpage">965</infon><infon key="lpage">72</infon><infon key="name_0">surname:Terwilliger;given-names:T. C.</infon><infon key="pub-id_pmid">10944333</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">56</infon><infon key="year">2000</infon><offset>30337</offset><text>Maximum-likelihood density modification</text></passage><passage><infon key="fpage">905</infon><infon key="lpage">21</infon><infon key="name_0">surname:Brünger;given-names:A. T.</infon><infon key="pub-id_pmid">9757107</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">54</infon><infon key="year">1998</infon><offset>30377</offset><text>Crystallography &amp; NMR system: A new software suite for macromolecular structure determination</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Lohkamp;given-names:B.</infon><infon key="name_2">surname:Scott;given-names:W. G.</infon><infon key="name_3">surname:Cowtan;given-names:K.</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>30471</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">240</infon><infon key="lpage">55</infon><infon key="name_0">surname:Murshudov;given-names:G. N.</infon><infon key="name_1">surname:Vagin;given-names:A. A.</infon><infon key="name_2">surname:Dodson;given-names:E. J.</infon><infon key="pub-id_pmid">15299926</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">53</infon><infon key="year">1997</infon><offset>30504</offset><text>Refinement of macromolecular structures by the maximum-likelihood method</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">42</infon><infon key="name_0">surname:Winn;given-names:M. D.</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>30577</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="fpage">277</infon><infon key="lpage">93</infon><infon key="name_0">surname:Delaglio;given-names:F.</infon><infon key="pub-id_pmid">8520220</infon><infon key="section_type">REF</infon><infon key="source">J Biomol NMR</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">1995</infon><offset>30629</offset><text>NMRPipe: a multidimensional spectral processing system based on UNIX pipes</text></passage><passage><infon key="fpage">353</infon><infon key="lpage">78</infon><infon key="name_0">surname:Guntert;given-names:P.</infon><infon key="pub-id_pmid">15318003</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol Biol</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2004</infon><offset>30704</offset><text>Automated NMR structure calculation with CYANA</text></passage><passage><infon key="fpage">289</infon><infon key="lpage">302</infon><infon key="name_0">surname:Cornilescu;given-names:G.</infon><infon key="name_1">surname:Delaglio;given-names:F.</infon><infon key="name_2">surname:Bax;given-names:A.</infon><infon key="pub-id_pmid">10212987</infon><infon key="section_type">REF</infon><infon key="source">J Biomol NMR</infon><infon key="type">ref</infon><infon key="volume">13</infon><infon key="year">1999</infon><offset>30751</offset><text>Protein backbone angle restraints from searching a database for chemical shift and sequence homology</text></passage><passage><infon key="fpage">2913</infon><infon key="lpage">4</infon><infon key="name_0">surname:Liang;given-names:S.</infon><infon key="name_1">surname:Zheng;given-names:D.</infon><infon key="name_2">surname:Zhang;given-names:C.</infon><infon key="name_3">surname:Standley;given-names:D. M.</infon><infon key="pub-id_pmid">21873640</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">27</infon><infon key="year">2011</infon><offset>30852</offset><text>Fast and accurate prediction of protein side-chain conformations</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>30917</offset><text>Author Contributions F.I. supervised the overall project. M.Y., T.T., Y.E., D.M.S., O.T., S.A. and F.I. designed the research; M.Y. and T.T. performed the research; M.Y., T.T., N.N.N., H.K., K.Y., D.M.S. and F.I. analyzed the data; and M.Y., N.N.N., H.K., K.Y., D.M.S. and F.I. wrote the paper. All authors reviewed the manuscript.</text></passage><passage><infon key="file">srep22324-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>31249</offset><text>Structural and functional analyses of Regnase-1.</text></passage><passage><infon key="file">srep22324-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31298</offset><text>(a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. Catalytic Asp residues were shown in sticks. (d) Solution structure of the ZF domain. Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. (e) Solution structure of the CTD. All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. (g) Binding of Regnase-1 and IL-6 mRNA was plotted. The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1.</text></passage><passage><infon key="file">srep22324-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32262</offset><text>Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1.</text></passage><passage><infon key="file">srep22324-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32360</offset><text>(a) Gel filtration analyses of the PIN domain. Elution volumes of the standard marker proteins were indicated by arrows at the upper part. (b) Dimer structure of the PIN domain. Two PIN molecules in the crystal were colored white and green, respectively. Catalytic residues and mutated residues were shown in sticks. Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed.</text></passage><passage><infon key="file">srep22324-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32894</offset><text>Domain-domain interaction between the NTD and the PIN domain.</text></passage><passage><infon key="file">srep22324-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32956</offset><text>(a) NMR analyses of the NTD-binding to the PIN domain. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). Pro and the residues without analysis were colored black and gray, respectively. (b) NMR analyses of the PIN-binding to the NTD. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. S62 was colored gray and excluded from the analysis, due to low signal intensity. (c) Docking model of the NTD and the PIN domain. The NTD and the PIN domain are shown in cyan and white, respectively. Residues in close proximity (&lt;5 Å) to each other in the docking structure were colored yellow. Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled.</text></passage><passage><infon key="file">srep22324-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33942</offset><text>Critical residues in the PIN domain for the RNase activity of Regnase-1.</text></passage><passage><infon key="file">srep22324-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34015</offset><text>(a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. The results indicate mean ± SD of four independent experiments. (b)
+In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow.</text></passage><passage><infon key="file">srep22324-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>34541</offset><text>Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity.</text></passage><passage><infon key="file">srep22324-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34666</offset><text>(a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN.</text></passage><passage><infon key="file">srep22324-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35284</offset><text>Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions.</text></passage></document></collection>

raw_BioC_XML/PMC4832331_raw.xml

@@ -0,0 +1,99 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4832331</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep24601</infon><infon key="article-id_pii">srep24601</infon><infon key="article-id_pmc">4832331</infon><infon key="article-id_pmid">27080013</infon><infon key="elocation-id">24601</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0
+International License. The images or other third party material in this article are
+included in the article’s Creative Commons license, unless indicated
+otherwise in the credit line; if the material is not included under the Creative
+Commons license, users will need to obtain permission from the license holder to
+reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Kandiah;given-names:Eaazhisai</infon><infon key="name_1">surname:Carriel;given-names:Diego</infon><infon key="name_10">surname:Gutsche;given-names:Irina</infon><infon key="name_2">surname:Perard;given-names:Julien</infon><infon key="name_3">surname:Malet;given-names:Hélène</infon><infon key="name_4">surname:Bacia;given-names:Maria</infon><infon key="name_5">surname:Liu;given-names:Kaiyin</infon><infon key="name_6">surname:Chan;given-names:Sze W. S.</infon><infon key="name_7">surname:Houry;given-names:Walid A.</infon><infon key="name_8">surname:Ollagnier de Choudens;given-names:Sandrine</infon><infon key="name_9">surname:Elsen;given-names:Sylvie</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>136</offset><text>The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1452</offset><text>Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach. Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment. These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2662</offset><text>Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4295</offset><text>The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4931</offset><text>Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6005</offset><text>In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. Moreover, this classification perfectly agrees with the genetic environment of the lysine decarboxylase genes. This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8130</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8153</offset><text>CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8208</offset><text>In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9121</offset><text>The wing domains as a stable anchor at the center of the double-ring</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9190</offset><text>As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10525</offset><text>The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10629</offset><text>Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). This interface is formed essentially by the core domains with some contribution of the CTDs. The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. Therefore we restrict our analysis to secondary structure elements. In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13132</offset><text>Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13228</offset><text>An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance. The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14916</offset><text>Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15003</offset><text>Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. Indeed, all CTDs have very similar structures (RMSDmin &lt;1 Å). Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15836</offset><text>The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15940</offset><text>In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. However, at the level of this structural element the two proteins are actually surpisingly similar. Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17457</offset><text>The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17587</offset><text>Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. Importantly, most of the amino acid differences between the two enzymes are located in this very region. Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). This procedure yielded several unexpected and exciting results. First of all, consensus sequence for the entire lysine decarboxylase family was derived. Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7). Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>22628</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22636</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22672</offset><text>LdcI and LdcC were expressed and purified as described from an E. coli strain that cannot produce ppGpp (MG1655 ΔrelA ΔspoT strain). LdcI was stored in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 6.8 (buffer A) and LdcC in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 7.5 (buffer B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22993</offset><text>Chimeric LdcIC and LdcCI were constructed, expressed and purified as follows. The chimeras were designed by exchange, between LdcI and LdcC, of residues from 631 to 640 and from 697 to the C-terminus, corresponding to the regions around the two strands of the C-terminal β-sheet (Fig. 5B,C), while leaving the rest of the sequence unaltered. The synthetic ldcIC and ldcCI genes (2148 bp and 2154 bp respectively), provided within a pUC57 vector (GenScript) were subcloned into pET-TEV vector based on pET-28a (Invitrogen) containing an N-terminal TEV-cleavable 6x-His-Tag. Proteins were expressed in Rosetta 2 (DE3) cells (Novagen) in LB medium supplemented with kanamycin and chloramphenicol at 37 °C, upon induction with 0.5 mM IPTG at 18 °C. Cells were harvested by centrifugation, the pellet resuspended in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with Complete EDTA free (Roche) and 0.1 mM PMSF (Sigma), and disrupted by sonication at 4 °C. After centrifugation at 75000 g at 4 °C for 20 min, the supernatant was loaded on a Ni-NTA column. The eluted protein-containing fractions were pooled and the His-Tag removed by incubation with the TEV protease at 1/100 ratio and an extensive dialysis in a 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, pH 8 buffer. After a second dialysis in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with 10 mM imidazole, the sample was loaded on a Ni-NTA column in the same buffer, which allowed to separate the TEV protease and LdcCI/LdcIC. Finally, the pure proteins were obtained by size exclusion chromatography on a Superdex-S200 column in buffer A.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24652</offset><text>LdcIa -cryoEM data collection and 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24704</offset><text>LdcI was prepared at 2 mg/ml in a buffer containing 25 mM MES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 6.2. 3 μl of sample were applied to glow-discharged quantifoil grids 300 mesh 2/1 (Quantifoil Micro Tools GmbH, Germany), excess solution was blotted during 2.5 s with a Vitrobot (FEI) and the grid frozen in liquid ethane. Data collection was performed on a FEI Polara microscope operated at 300 kV under low dose conditions. Micrographs were recorded on Kodak SO-163 film at 59,000 magnification, with defocus ranging from 0.6 to 4.9 μm. Films were digitized on a Zeiss scanner (Photoscan) at a step size of 7 μm giving a pixel size of 1.186 Å. The contrast transfer function (CTF) for each micrograph was determined with CTFFIND3.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25462</offset><text>Initially ~2500 particles of 256 × 256 pixels were extracted manually, binned 4 times and subjected to one round of multivariate statistical analysis and classification using IMAGIC. Representative class averages corresponding to projections in different orientations were used as input for an ab-initio 3D reconstruction by RICOserver (rico.ibs.fr/). The resulting 3D reconstruction was refined using RELION, which yielded an 18 Å resolution map. Using projections of this model as a template, particles of size 256 × 256 pixels were semi-automatically selected from all the micrographs using the Fast Projection Matching (FPM) algorithm. The resulting dataset of ~46000 particles was processed in RELION with the previous map as an initial model and with a full CTF correction after the first peak. The final map comprised 44207 particles with a resolution of 7.4 Å as per the gold-standard FSC = 0.143 criterion. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.1 Å (Fig. S3, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26571</offset><text>LdcC - cryoEM data collection and 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26623</offset><text>LdcC was prepared at 2 mg/ml in a buffer containing 25 mM HEPES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 7.2. Grids were prepared and sample imaged as LdcIa. Data were processed essentially as LdcIa described above. Briefly, an initial ~20 Å resolution model was generated by angular reconstitution after manual picking of circa 3000 particles from the first micrographs, filtered to 60 Å resolution, refined with RELION and used for a semi-automatic selection with FPM. The dataset was processed in RELION with a full CTF correction to yield a final reconstruction comprising 61000 particles. The map was sharpened with Relion postprocessing, using a soft mask and automated B-factor estimation (−690 Å2), yielding a map at 5.5 Å resolution (Fig. S1, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27400</offset><text>LdcI-LARA - 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27430</offset><text>In our first study, the dataset was processed in SPIDER and the CTF correction involved a simple phase-flipping. Therefore, for consistency with the present work, we revisited the dataset and processed it in RELION with a full CTF correction after the first peak. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.2 Å (Fig. S2). This reconstruction is of a slightly better quality in terms of the continuity of the internal density. Therefore we used this improved map for fitting of the atomic model and further analysis (Fig. S2, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28073</offset><text>Additional image processing</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28101</offset><text>As a crosscheck, each data set was also refined either from other initial models: a “featureless donut” with approximate dimensions of the decamer, and low pass-filtered reconstructions from the two other data sets (i.e. the LdcC reconstruction was used as a model for the LdcIa and LdcI-LARA data sets, etc). All refinements converged to the same solutions independently of the starting model. Local resolution of all maps was determined with ResMap.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28557</offset><text>LdcCI and LdcIC chimeras —negative stain EM and 2D image analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28625</offset><text>0.4 mg/ml of RavA (in a 20 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT, 5% glycerol, pH 6.8 buffer) was mixed with 0.3 mg/ml of either LdcI, LdcC, LdcCI or LdcIC in the presence of 2 mM ADP and 10 mM MgCl2 in a buffer containing 20 mM Hepes and 150 mM NaCl at pH 7.4. After 10 minutes incubation at room temperature, 3 μl of mixture were applied to the clear side of the carbon on a carbon-mica interface and negatively stained with 2% uranyl acetate. Images were recorded with a JEOL 1200 EX II microscope at 100 kV at a nominal magnification of 15000 on a CCD camera yielding a pixel size of 4.667 Å. No complexes between RavA and LdcC or LdcIC could be observed, whereas the LdcCI-RavA preparation manifested cage-like particles similar to the previously published LdcI-RavA, but also unbound RavA and LdcCI, which implies that the affinity of RavA to the LdcCI chimera is lower than its affinity to the native LdcI. 1260 particles of 96 × 96 pixels were extracted interactively from several micrographs. 2D centering, multivariate statistical analysis and classification were performed using IMAGIC. Class-averages similar to the cage-like LdcI-RavA complex were used as references for multi-reference alignment followed by multivariate statistical analysis and classification.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29946</offset><text>Fitting of atomic models into cryoEM maps</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29988</offset><text>A homology model of LdcC was obtained using the atomic coordinates of the LdcI monomer (3N75) as the template in SWISS-MODEL server. The RMSD between the template and the resulting model was 0.26 Å. The LdcC model was then fitted as a rigid body into the LdcC cryoEM map using the fit-in-map module of UCSF Chimera. This rigid fit indicated movements of several parts of the protein. Therefore, the density corresponding to one LdcC monomer was extracted and flexible fitting was performed using IMODFIT at 8 Å resolution. This monomeric model was then docked into the decameric cryoEM map with URO and its graphical version VEDA that use symmetry information for fitting in Fourier space. The Cα RMSDmin between the initial model of the LdcC monomer and the final IMODFIT LdcC model is 1.2 Å. In the case of LdcIa, the density corresponding to an individual monomer was extracted and the fit performed similarly to the one described above, with the final model of the decameric particle obtained with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. For LdcI-LARA, the density accounting for one LdcI monomer bound to a LARA domain was extracted and further separated into individual densities corresponding to LdcI and to LARA. The fit of LdcI was performed as for LdcC and LdcIa, while the crystal structure of LARA was docked into the monomeric LdcI-LARA map as a rigid body using SITUS. The resulting pseudoatomic models were used to create the final model of the LdcI-LARA decamer with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. A brief summary of relevant parameters is provided in Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31699</offset><text>Sequence analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31717</offset><text>Out of a non-exhaustive list of 50 species of Enterobacteriaceae (Table S3), 22 were found to contain genes annotated as ldcI or ldcC and containing the ravA-viaA operon (Table S4). An analysis using MUSCLE with default parameters showed that these genes share more than 65% identity. To verify annotation of these genes, we compared their genetic environment with that of E. coli ldcI and ldcC. Indeed, in E. coli the ldcI gene is in operon with the cadB gene encoding a lysine-cadaverine antiporter, whereas the ldcC gene is present between the accA gene (encoding an acetyl-CoA carboxylase alpha subunit carboxyltransferase) and the yaeR gene (coding for an unknown protein belonging to the family of Glyoxalase/Dioxygenase/Bleomycin resistance proteins). Compared with this genetic environment, the annotation of several ldcI and ldcC genes in enterobacteria was found to be inconsistent (Table S4). For example, several strains contain genes annotated as ldcC in the genetic background of ldcI and vice versa, as in the case of Salmonella enterica and Trabulsiella guamensi. Furthermore, the gene with an “ldcC-like” environment was found to be annotated as cadA in particular strains of Citrobacter freundii, Cronobacter sakazakii, Enterobacter cloacae subsp. Cloaca, Erwinia amylovora, Pantoea agglomerans, Rahnella aquatilis, Shigella dysenteriae, and Yersinia enterocolitica subsp. enterocolitica, whereas in Hafnia alvei, Kluyvera ascorbata, and Serratia marcescens subsp. marcescens, the gene with an “ldcI-like” environment was found to be annotated as ldcC. In addition, as far as the genetic environment is concerned, Plesiomonas appears to have two ldc genes with the organization of the E. coli ldcI (operon cadA-cadB). Consequently, we corrected for gene annotation consistent with the genetic environment and made multiple sequence alignments using version 8.0.1 of the CLC Genomics Workbench software. A phylogenetic tree was generated based on Maximum Likelihood and following the Neighbor-Joining method with the WAG protein substitution model. The reliability of the generated phylogenetic tree was assessed by bootstrap analysis. The presented unrooted phylogenetic tree shows the nodes that are reliable over 95% (Fig. 6A). Remarkably, the multiple sequence alignment and the resulting phylogenetic tree clearly grouped together all sequences annotated as ldcI on the one side, and all sequences annotated as ldcC on the other side. Thus, we conclude that all modifications in gene annotations that we introduced for the sake of consistency with the genetic environment are perfectly corroborated by the multiple sequence alignment and the phylogenetic analysis. Since the regulation of the lysine decarboxylase gene (i.e. inducible or constitutive) cannot be assessed by this analysis, we call the resulting groups “ldcI-like” and “ldcC-like” as referred to the E. coli enzymes, and make a parallel between the membership in a given group and the ability of the protein to form a cage complex with RavA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34762</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34785</offset><text>Accession codes: CryoEM maps and corresponding fitted atomic structures (main chain atoms) have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, respectively, with accession codes EMD-3205 and 5FKZ for LdcC, EMD-3204 and 5FKX for LdcIa and EMD-3206 and 5FL2 for LdcI-LARA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35084</offset><text>How to cite this article: Kandiah, E. et al. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. Sci. Rep. 6, 24601; doi: 10.1038/srep24601 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>35317</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">436</infon><infon key="lpage">447</infon><infon key="name_0">surname:Christen;given-names:P.</infon><infon key="name_1">surname:Mehta;given-names:P. K.</infon><infon key="section_type">REF</infon><infon key="source">Chem. Rec.
+N. Y. N</infon><infon key="type">ref</infon><infon key="volume">1</infon><infon key="year">2001</infon><offset>35340</offset><text>From cofactor to enzymes. The molecular evolution of pyridoxal-5′-phosphate-dependent enzymes</text></passage><passage><infon key="fpage">383</infon><infon key="lpage">415</infon><infon key="name_0">surname:Eliot;given-names:A. C.</infon><infon key="name_1">surname:Kirsch;given-names:J. F.</infon><infon key="pub-id_pmid">15189147</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev. Biochem.</infon><infon key="type">ref</infon><infon key="volume">73</infon><infon key="year">2004</infon><offset>35436</offset><text>Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations</text></passage><passage><infon key="fpage">301</infon><infon key="lpage">314</infon><infon key="name_0">surname:Zhao;given-names:B.</infon><infon key="name_1">surname:Houry;given-names:W. A.</infon><infon key="section_type">REF</infon><infon key="source">Biochem. Cell Biol. Biochim. Biol. Cell.</infon><infon key="type">ref</infon><infon key="volume">88</infon><infon key="year">2010</infon><offset>35522</offset><text>Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival</text></passage><passage><infon key="fpage">65</infon><infon key="lpage">81</infon><infon key="name_0">surname:Kanjee;given-names:U.</infon><infon key="name_1">surname:Houry;given-names:W. A.</infon><infon key="pub-id_pmid">23701194</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev.
+Microbiol.</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2013</infon><offset>35609</offset><text>Mechanisms of acid resistance in Escherichia coli</text></passage><passage><infon key="fpage">e22397</infon><infon key="name_0">surname:Viala;given-names:J. P. M.</infon><infon key="pub-id_pmid">21799843</infon><infon key="section_type">REF</infon><infon key="source">PloS One</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2011</infon><offset>35659</offset><text>Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella</text></passage><passage><infon key="fpage">600</infon><infon key="lpage">618</infon><infon key="name_0">surname:Gale;given-names:E. F.</infon><infon key="name_1">surname:Epps;given-names:H. M.</infon><infon key="pub-id_pmid">16747564</infon><infon key="section_type">REF</infon><infon key="source">Biochem.
+J.</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">1942</infon><offset>35754</offset><text>The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced</text></passage><passage><infon key="fpage">232</infon><infon key="lpage">242</infon><infon key="name_0">surname:Gale;given-names:E. F.</infon><infon key="name_1">surname:Epps;given-names:H. M.</infon><infon key="pub-id_pmid">16747785</infon><infon key="section_type">REF</infon><infon key="source">Biochem. J.</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">1944</infon><offset>35946</offset><text>Studies on bacterial amino-acid decarboxylases: 1. l(+)-lysine decarboxylase</text></passage><passage><infon key="fpage">836</infon><infon key="lpage">849</infon><infon key="name_0">surname:Merrell;given-names:D. S.</infon><infon key="name_1">surname:Camilli;given-names:A.</infon><infon key="pub-id_pmid">10564522</infon><infon key="section_type">REF</infon><infon key="source">Mol. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">34</infon><infon key="year">1999</infon><offset>36023</offset><text>The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance</text></passage><passage><infon key="fpage">8586</infon><infon key="lpage">8592</infon><infon key="name_0">surname:Kim;given-names:J.-S.</infon><infon key="name_1">surname:Choi;given-names:S. H.</infon><infon key="name_2">surname:Lee;given-names:J.
+K.</infon><infon key="pub-id_pmid">17012399</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">188</infon><infon key="year">2006</infon><offset>36119</offset><text>Lysine decarboxylase expression by Vibrio vulnificus is induced by SoxR in response to superoxide stress</text></passage><passage><infon key="fpage">928</infon><infon key="lpage">933</infon><infon key="name_0">surname:Bower;given-names:J. M.</infon><infon key="name_1">surname:Mulvey;given-names:M. A.</infon><infon key="pub-id_pmid">16428396</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">188</infon><infon key="year">2006</infon><offset>36224</offset><text>Polyamine-mediated resistance of uropathogenic Escherichia coli to nitrosative stress</text></passage><passage><infon key="fpage">2104</infon><infon key="lpage">2112</infon><infon key="name_0">surname:Bower;given-names:J. M.</infon><infon key="name_1">surname:Gordon-Raagas;given-names:H. B.</infon><infon key="name_2">surname:Mulvey;given-names:M. A.</infon><infon key="pub-id_pmid">19255192</infon><infon key="section_type">REF</infon><infon key="source">Infect. Immun.</infon><infon key="type">ref</infon><infon key="volume">77</infon><infon key="year">2009</infon><offset>36310</offset><text>Conditioning of uropathogenic Escherichia coli for enhanced colonization of host</text></passage><passage><infon key="fpage">575</infon><infon key="lpage">579</infon><infon key="name_0">surname:Akhova;given-names:A. V.</infon><infon key="name_1">surname:Tkachenko;given-names:A. G.</infon><infon key="section_type">REF</infon><infon key="source">Microbiology</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">2009</infon><offset>36391</offset><text>Lysine Decarboxylase Activity as a Factor of Fluoroquinolone Resistance in Escherichia coli</text></passage><passage><infon key="fpage">1267</infon><infon key="lpage">1270</infon><infon key="name_0">surname:Kikuchi;given-names:Y.</infon><infon key="name_1">surname:Kurahashi;given-names:O.</infon><infon key="name_2">surname:Nagano;given-names:T.</infon><infon key="name_3">surname:Kamio;given-names:Y.</infon><infon key="pub-id_pmid">9692215</infon><infon key="section_type">REF</infon><infon key="source">Biosci. Biotechnol. Biochem.</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">1998</infon><offset>36483</offset><text>RpoS-dependent expression of the second lysine decarboxylase gene in Escherichia coli</text></passage><passage><infon key="fpage">13</infon><infon key="lpage">19</infon><infon key="name_0">surname:Samartzidou;given-names:H.</infon><infon key="name_1">surname:Mehrazin;given-names:M.</infon><infon key="name_2">surname:Xu;given-names:Z.</infon><infon key="name_3">surname:Benedik;given-names:M.
+J.</infon><infon key="name_4">surname:Delcour;given-names:A.
+H.</infon><infon key="pub-id_pmid">12486035</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">185</infon><infon key="year">2003</infon><offset>36569</offset><text>Cadaverine inhibition of porin plays a role in cell survival at acidic pH</text></passage><passage><infon key="fpage">330</infon><infon key="lpage">334</infon><infon key="name_0">surname:Bekhit;given-names:A.</infon><infon key="name_1">surname:Fukamachi;given-names:T.</infon><infon key="name_2">surname:Saito;given-names:H.</infon><infon key="name_3">surname:Kobayashi;given-names:H.</infon><infon key="pub-id_pmid">21372380</infon><infon key="section_type">REF</infon><infon key="source">Biol. Pharm. Bull.</infon><infon key="type">ref</infon><infon key="volume">34</infon><infon key="year">2011</infon><offset>36643</offset><text>The role of OmpC and OmpF in acidic resistance in Escherichia coli</text></passage><passage><infon key="fpage">1042</infon><infon key="lpage">1049</infon><infon key="name_0">surname:Tkachenko;given-names:A. G.</infon><infon key="name_1">surname:Pozhidaeva;given-names:O. N.</infon><infon key="name_2">surname:Shumkov;given-names:M. S.</infon><infon key="section_type">REF</infon><infon key="source">Biochem.
+Biokhimiia</infon><infon key="type">ref</infon><infon key="volume">71</infon><infon key="year">2006</infon><offset>36710</offset><text>Role of polyamines in formation of multiple antibiotic resistance of Escherichia coli under stress conditions</text></passage><passage><infon key="fpage">931</infon><infon key="lpage">944</infon><infon key="name_0">surname:Kanjee;given-names:U.</infon><infon key="pub-id_pmid">21278708</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2011</infon><offset>36820</offset><text>Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase</text></passage><passage><infon key="fpage">662</infon><infon key="lpage">670</infon><infon key="name_0">surname:Sabo;given-names:D. L.</infon><infon key="name_1">surname:Boeker;given-names:E. A.</infon><infon key="name_2">surname:Byers;given-names:B.</infon><infon key="name_3">surname:Waron;given-names:H.</infon><infon key="name_4">surname:Fischer;given-names:E. H.</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry (Mosc.)</infon><infon key="type">ref</infon><infon key="volume">13</infon><infon key="year">1974</infon><offset>36939</offset><text>Purification and physical properties of inducible Escherichia coli lysine decarboxylase</text></passage><passage><infon key="fpage">1532</infon><infon key="lpage">1546</infon><infon key="name_0">surname:Snider;given-names:J.</infon><infon key="pub-id_pmid">16301313</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">281</infon><infon key="year">2006</infon><offset>37027</offset><text>Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase</text></passage><passage><infon key="fpage">211</infon><infon key="lpage">221</infon><infon key="name_0">surname:Wong;given-names:K. S.</infon><infon key="name_1">surname:Houry;given-names:W. A.</infon><infon key="pub-id_pmid">22491058</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">179</infon><infon key="year">2012</infon><offset>37139</offset><text>Novel structural and functional insights into the MoxR family of AAA+ ATPases</text></passage><passage><infon key="fpage">22499</infon><infon key="lpage">22504</infon><infon key="name_0">surname:El Bakkouri;given-names:M.</infon><infon key="pub-id_pmid">21148420</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">107</infon><infon key="year">2010</infon><offset>37217</offset><text>Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity</text></passage><passage><infon key="fpage">e85529</infon><infon key="name_0">surname:Wong;given-names:K. S.</infon><infon key="pub-id_pmid">24454883</infon><infon key="section_type">REF</infon><infon key="source">PloS
+One</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2014</infon><offset>37358</offset><text>The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli</text></passage><passage><infon key="fpage">e03653</infon><infon key="name_0">surname:Malet;given-names:H.</infon><infon key="pub-id_pmid">25097238</infon><infon key="section_type">REF</infon><infon key="source">eLife</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2014</infon><offset>37472</offset><text>Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins</text></passage><passage><infon key="fpage">167</infon><infon key="lpage">172</infon><infon key="name_0">surname:Yamamoto;given-names:Y.</infon><infon key="name_1">surname:Miwa;given-names:Y.</infon><infon key="name_2">surname:Miyoshi;given-names:K.</infon><infon key="name_3">surname:Furuyama;given-names:J.</infon><infon key="name_4">surname:Ohmori;given-names:H.</infon><infon key="pub-id_pmid">9339543</infon><infon key="section_type">REF</infon><infon key="source">Genes Genet. Syst.</infon><infon key="type">ref</infon><infon key="volume">72</infon><infon key="year">1997</infon><offset>37560</offset><text>The Escherichia coli ldcC gene encodes another lysine decarboxylase, probably a constitutive enzyme</text></passage><passage><infon key="fpage">857</infon><infon key="lpage">876</infon><infon key="name_0">surname:Käck;given-names:H.</infon><infon key="name_1">surname:Sandmark;given-names:J.</infon><infon key="name_2">surname:Gibson;given-names:K.</infon><infon key="name_3">surname:Schneider;given-names:G.</infon><infon key="name_4">surname:Lindqvist;given-names:Y.</infon><infon key="pub-id_pmid">10452893</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">291</infon><infon key="year">1999</infon><offset>37660</offset><text>Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5′-phosphate-dependent enzymes</text></passage><passage><infon key="fpage">9388</infon><infon key="lpage">9398</infon><infon key="name_0">surname:Kanjee;given-names:U.</infon><infon key="name_1">surname:Gutsche;given-names:I.</infon><infon key="name_2">surname:Ramachandran;given-names:S.</infon><infon key="name_3">surname:Houry;given-names:W.
+A.</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry
+(Mosc.)</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2011</infon><offset>37792</offset><text>The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition</text></passage><passage><infon key="fpage">863</infon><infon key="lpage">871</infon><infon key="name_0">surname:Erhardt;given-names:H.</infon><infon key="pub-id_pmid">22063474</infon><infon key="section_type">REF</infon><infon key="source">Biochim. Biophys. Acta</infon><infon key="type">ref</infon><infon key="volume">1817</infon><infon key="year">2012</infon><offset>37915</offset><text>Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli</text></passage><passage><infon key="fpage">1176</infon><infon key="lpage">1184</infon><infon key="name_0">surname:Lohinai;given-names:Z.</infon><infon key="pub-id_pmid">26110450</infon><infon key="section_type">REF</infon><infon key="source">J. Periodontol.</infon><infon key="type">ref</infon><infon key="volume">86</infon><infon key="year">2015</infon><offset>38059</offset><text>Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation</text></passage><passage><infon key="fpage">6706</infon><infon key="lpage">6712</infon><infon key="name_0">surname:Peters;given-names:J. L.</infon><infon key="pub-id_pmid">22975025</infon><infon key="section_type">REF</infon><infon key="source">Vaccine</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2012</infon><offset>38143</offset><text>Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development</text></passage><passage><infon key="fpage">1048</infon><infon key="lpage">1056</infon><infon key="name_0">surname:Lohinai;given-names:Z.</infon><infon key="pub-id_pmid">22141361</infon><infon key="section_type">REF</infon><infon key="source">J. Periodontol.</infon><infon key="type">ref</infon><infon key="volume">83</infon><infon key="year">2012</infon><offset>38250</offset><text>Bacterial lysine decarboxylase influences human dental biofilm lysine content, biofilm accumulation, and subclinical gingival inflammation</text></passage><passage><infon key="fpage">1108</infon><infon key="lpage">1113</infon><infon key="name_0">surname:Kim;given-names:H. J.</infon><infon key="pub-id_pmid">25674800</infon><infon key="section_type">REF</infon><infon key="source">J. Microbiol. Biotechnol</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2015</infon><offset>38389</offset><text>Optimization of Direct Lysine Decarboxylase Biotransformation for Cadaverine Production with Whole-Cell Biocatalysts at High Lysine Concentration</text></passage><passage><infon key="fpage">799</infon><infon key="lpage">806</infon><infon key="name_0">surname:Ma;given-names:W.</infon><infon key="pub-id_pmid">25515797</infon><infon key="section_type">REF</infon><infon key="source">Biotechnol. Lett.</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">2015</infon><offset>38535</offset><text>Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB</text></passage><passage><infon key="fpage">965</infon><infon key="lpage">972</infon><infon key="name_0">surname:Li;given-names:N.</infon><infon key="name_1">surname:Chou;given-names:H.</infon><infon key="name_2">surname:Yu;given-names:L.</infon><infon key="name_3">surname:Xu;given-names:Y.</infon><infon key="section_type">REF</infon><infon key="source">Biotechnol. Bioprocess Eng.</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2014</infon><offset>38647</offset><text>Cadaverine production by heterologous expression of Klebsiella oxytoca lysine decarboxylase</text></passage><passage><infon key="fpage">129</infon><infon key="lpage">228</infon><infon key="name_0">surname:Dubochet;given-names:J.</infon><infon key="pub-id_pmid">3043536</infon><infon key="section_type">REF</infon><infon key="source">Q. Rev.
+Biophys.</infon><infon key="type">ref</infon><infon key="volume">21</infon><infon key="year">1988</infon><offset>38739</offset><text>Cryo-electron microscopy of vitrified specimens</text></passage><passage><infon key="fpage">334</infon><infon key="lpage">347</infon><infon key="name_0">surname:Mindell;given-names:J. A.</infon><infon key="name_1">surname:Grigorieff;given-names:N.</infon><infon key="pub-id_pmid">12781660</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">142</infon><infon key="year">2003</infon><offset>38787</offset><text>Accurate determination of local defocus and specimen tilt in electron microscopy</text></passage><passage><infon key="fpage">17</infon><infon key="lpage">24</infon><infon key="name_0">surname:van Heel;given-names:M.</infon><infon key="name_1">surname:Harauz;given-names:G.</infon><infon key="name_2">surname:Orlova;given-names:E.
+V.</infon><infon key="name_3">surname:Schmidt;given-names:R.</infon><infon key="name_4">surname:Schatz;given-names:M.</infon><infon key="pub-id_pmid">8742718</infon><infon key="section_type">REF</infon><infon key="source">J.
+Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">116</infon><infon key="year">1996</infon><offset>38868</offset><text>A new generation of the IMAGIC image processing system</text></passage><passage><infon key="fpage">13</infon><infon key="lpage">23</infon><infon key="name_0">surname:Navaza;given-names:J.</infon><infon key="pub-id_pmid">14643206</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">144</infon><infon key="year">2003</infon><offset>38923</offset><text>On the three-dimensional reconstruction of icosahedral particles</text></passage><passage><infon key="fpage">519</infon><infon key="lpage">530</infon><infon key="name_0">surname:Scheres;given-names:S. H. W.</infon><infon key="pub-id_pmid">23000701</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">180</infon><infon key="year">2012</infon><offset>38988</offset><text>RELION: implementation of a Bayesian approach to cryo-EM structure determination</text></passage><passage><infon key="fpage">253</infon><infon key="lpage">260</infon><infon key="name_0">surname:Estrozi;given-names:L. F.</infon><infon key="name_1">surname:Navaza;given-names:J.</infon><infon key="pub-id_pmid">20599509</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">172</infon><infon key="year">2010</infon><offset>39069</offset><text>Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way</text></passage><passage><infon key="fpage">853</infon><infon key="lpage">854</infon><infon key="name_0">surname:Scheres;given-names:S. H. W.</infon><infon key="name_1">surname:Chen;given-names:S.</infon><infon key="pub-id_pmid">22842542</infon><infon key="section_type">REF</infon><infon key="source">Nat. Methods</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2012</infon><offset>39166</offset><text>Prevention of overfitting in cryo-EM structure determination</text></passage><passage><infon key="fpage">170</infon><infon key="lpage">175</infon><infon key="name_0">surname:Fernández;given-names:J. J.</infon><infon key="name_1">surname:Luque;given-names:D.</infon><infon key="name_2">surname:Castón;given-names:J. R.</infon><infon key="name_3">surname:Carrascosa;given-names:J. L.</infon><infon key="pub-id_pmid">18614378</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">164</infon><infon key="year">2008</infon><offset>39227</offset><text>Sharpening high resolution information in single particle electron cryomicroscopy</text></passage><passage><infon key="fpage">63</infon><infon key="lpage">65</infon><infon key="name_0">surname:Kucukelbir;given-names:A.</infon><infon key="name_1">surname:Sigworth;given-names:F. J.</infon><infon key="name_2">surname:Tagare;given-names:H. D.</infon><infon key="pub-id_pmid">24213166</infon><infon key="section_type">REF</infon><infon key="source">Nat.
+Methods</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2014</infon><offset>39309</offset><text>Quantifying the local resolution of cryo-EM density maps</text></passage><passage><infon key="fpage">3381</infon><infon key="lpage">3385</infon><infon key="name_0">surname:Schwede;given-names:T.</infon><infon key="name_1">surname:Kopp;given-names:J.</infon><infon key="name_2">surname:Guex;given-names:N.</infon><infon key="name_3">surname:Peitsch;given-names:M. C.</infon><infon key="pub-id_pmid">12824332</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">31</infon><infon key="year">2003</infon><offset>39366</offset><text>SWISS-MODEL: An automated protein homology-modeling server</text></passage><passage><infon key="fpage">1605</infon><infon key="lpage">1612</infon><infon key="name_0">surname:Pettersen;given-names:E. F.</infon><infon key="pub-id_pmid">15264254</infon><infon key="section_type">REF</infon><infon key="source">J. Comput. Chem.</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2004</infon><offset>39425</offset><text>UCSF Chimera–a visualization system for exploratory research and analysis</text></passage><passage><infon key="fpage">261</infon><infon key="lpage">270</infon><infon key="name_0">surname:Lopéz-Blanco;given-names:J. R.</infon><infon key="name_1">surname:Chacón;given-names:P.</infon><infon key="pub-id_pmid">23999189</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">184</infon><infon key="year">2013</infon><offset>39501</offset><text>iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates</text></passage><passage><infon key="fpage">1820</infon><infon key="lpage">1825</infon><infon key="name_0">surname:Navaza;given-names:J.</infon><infon key="name_1">surname:Lepault;given-names:J.</infon><infon key="name_2">surname:Rey;given-names:F. A.</infon><infon key="name_3">surname:Alvarez-Rúa;given-names:C.</infon><infon key="name_4">surname:Borge;given-names:J.</infon><infon key="pub-id_pmid">12351826</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol.
+Crystallogr</infon><infon key="type">ref</infon><infon key="volume">58</infon><infon key="year">2002</infon><offset>39602</offset><text>On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation</text></passage><passage><infon key="fpage">651</infon><infon key="lpage">658</infon><infon key="name_0">surname:Siebert;given-names:X.</infon><infon key="name_1">surname:Navaza;given-names:J.</infon><infon key="pub-id_pmid">19564685</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol.
+Crystallogr</infon><infon key="type">ref</infon><infon key="volume">65</infon><infon key="year">2009</infon><offset>39701</offset><text>UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions</text></passage><passage><infon key="fpage">1792</infon><infon key="lpage">1797</infon><infon key="name_0">surname:Edgar;given-names:R. C.</infon><infon key="pub-id_pmid">15034147</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2004</infon><offset>39798</offset><text>MUSCLE: multiple sequence alignment with high accuracy and high throughput</text></passage><passage><infon key="fpage">691</infon><infon key="lpage">699</infon><infon key="name_0">surname:Whelan;given-names:S.</infon><infon key="name_1">surname:Goldman;given-names:N.</infon><infon key="pub-id_pmid">11319253</infon><infon key="section_type">REF</infon><infon key="source">Mol. Biol.
+Evol.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2001</infon><offset>39873</offset><text>A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>39995</offset><text>Author Contributions E.K., H.M. and I.G. carried out EM data collection with assistance of M.B. and analyzed the data. D.C. performed cloning, multiple sequence alignment and phylogenetic analysis under the direction of S.E. and I.G., J.P. cloned and purified chimeric proteins under the direction of S.O.C., K.L. and S.W.S.C. purified LdcI, LdcC and LARA under the direction of W.A.H., I.G. conceived and directed the studies and wrote the manuscript with input from E.K.</text></passage><passage><infon key="file">srep24601-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40468</offset><text>3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa.</text></passage><passage><infon key="file">srep24601-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40524</offset><text>(A,C,E) cryoEM map of the LdcC (A), LdcIa
+(C) and LdcI-LARA (E) decamers with one protomer in light
+grey. In the rest of the protomers, the wing, core and C-terminal domains
+are colored from light to dark in shades of green for LdcC (A), pink
+for LdcIa (C) and blue for LdcI in LdcI-LARA (E).
+In (E), the LARA domain density is shown in dark grey. Two monomers
+making a dimer are delineated. Scale bar 50 Å.
+(B,D,F) One protomer from the cryoEM map of the LdcC (B),
+LdcIa (D) and LdcI-LARA (F) in light grey with
+the pseudoatomic model represented as cartoons and colored as the densities
+in (A,C,E). Each domain is indicated for clarity. Scale bar
+50 Å. See also Figs S1 and S3.</text></passage><passage><infon key="file">srep24601-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41210</offset><text>Analysis of conformational rearrangements.</text></passage><passage><infon key="file">srep24601-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41253</offset><text>Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and
+LdcIa colored as in Fig. 1, and the
+crystal structure of LdcIi in shades of yellow. Only one of the
+two rings of the double toroid is shown for clarity. The dashed circle
+indicates the central region that remains virtually unchanged between all
+the structures, while the periphery undergoes visible movements. Scale bar
+50 Å.</text></passage><passage><infon key="file">srep24601-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41656</offset><text>Conformational rearrangements in the enzyme active site.</text></passage><passage><infon key="file">srep24601-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41713</offset><text>(A) LdcIi crystal structure, with one ring represented as a
+grey surface and the second as a cartoon. A monomer with its PLP cofactor is
+delineated. The PLP moieties of the cartoon ring are shown in red.
+(B) The LdcIi dimer extracted from the crystal structure
+of the decamer. One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer
+related by C2 symmetry is grey. The PLP is red. The active site is boxed.
+(C–F) Close-up views of the active site. The PLP
+moiety in red is from the LdcIi crystal structure. We did not
+attempt to model it in the cryoEM maps. The dimer interface is shown as a
+dashed line and the active site α-helices mentioned in the text
+are highlighted. (C) Compares LdcIi (yellow) and
+LdcIa (pink), (D) compares LdcIa (pink) and
+LdcI-LARA (blue), and (E) compares LdcIi (yellow),
+LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to
+show the progressive shift described in the text. (F) Shows the
+similarity between LdcIa and LdcC at the level of the secondary
+structure elements composing the active site. Colors are as in the other
+figures.</text></passage><passage><infon key="file">srep24601-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42813</offset><text>Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA
+binding.</text></passage><passage><infon key="file">srep24601-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42898</offset><text>(A–C) A slice through the pseudoatomic models of the LdcI
+monomers extracted from the superimposed decamers (Fig.
+2) The rectangle indicates the regions enlarged in
+(D–F). (A) compares LdcIi (yellow)
+and LdcIa (pink), (B) compares LdcIa (pink) and
+LdcI-LARA (blue), and (C) compares LdcIi (yellow),
+LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to
+show the progressive stretching described in the text. The cryoEM density of
+the LARA domain is represented as a grey surface to show the position of the
+binding site and the direction of the movement. (D–F)
+Inserts zooming at the CTD part in proximity of the LARA binding site. Loop
+regions are removed for a clearer visual comparison. An arrow indicates a
+swinging movement.</text></passage><passage><infon key="file">srep24601-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43641</offset><text>Analysis of the LdcIC and LdcCI chimeras.</text></passage><passage><infon key="file">srep24601-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43683</offset><text>(A) A slice through the pseudoatomic models of the LdcIa
+(purple) and LdcC (green) monomers extracted from the superimposed decamers
+(Fig. 2). (B) The C-terminal
+β-sheet in LdcIa and LdcC enlarged from
+(A,C) Exchanged primary sequences (capital letters) and
+their immediate vicinity (lower case letters) colored as in
+(A,B), with the corresponding secondary structure elements
+and the amino acid numbering shown. (D,E) A gallery of negative stain
+EM images of (D) the wild type LdcI-RavA cage and (E) the
+LdcCI-RavA cage-like particles. (F) Some representative class
+averages of the LdcCI-RavA cage-like particles. Scale bar
+20 nm.</text></passage><passage><infon key="file">srep24601-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44318</offset><text>Sequence analysis of enterobacterial lysine decarboxylases.</text></passage><passage><infon key="file">srep24601-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44378</offset><text>(A) Maximum likelihood tree with the
+“LdcC-like” and the
+“LdcI-like” groups highlighted in green and pink,
+respectively. Only nodes with higher values than 95% are shown (500
+replicates of the original dataset, see Methods for details). Scale bar
+indicates the average number of substitutions per site. (B) Analysis
+of consensus “LdcI-like” and
+“LdcC-like” sequences around the first and second
+C-terminal β-strands. The height of the bars and the letters
+representing the amino acids reflects the degree of conservation of each
+particular position is in the alignment. Amino acids are colored according
+to a polarity color scheme with hydrophobic residues in black, hydrophilic
+in green, acidic in red and basic in blue. Numbering as in E. coli.
+(C) Signature sequences of LdcI and LdcC in the C-terminal
+β-sheet. Polarity differences are highlighted. (D)
+Position and nature of these differences at the surface of the respective
+cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4.</text></passage></document></collection>