nils-herrmann/clean_scirepeval huggingface dataset:
Resolving the activation mechanism of the D99N antiterminator LicT protein.
Venue: Journal of structural biology

LicT is an antiterminator protein of the BglG family whose members are key players in the control of carbohydrate catabolism in bacteria. These antiterminators are generally composed of three modules, an N-terminal RNA-binding domain (CAT) followed by two homologous regulation modules (PRD1 and PRD2) that control the RNA binding activity of the effector domain via phosphorylation on conserved histidines. Although several structures of isolated domains of BglG-like proteins have been described, no structure containing CAT and at least one PRD simultaneously has yet been reported in an active state, precluding detailed understanding of signal transduction between modules. To fulfill this gap, we recently reported the complete NMR sequence assignment of a constitutively active mutant (D99N) CAT-PRD1*, which contains the effector domain and the first regulation domain of LicT. As a follow-up, we have determined and report here the 3D solution structure of this active, dimeric LicT construct (40 kDa). The structure reveals how the mutation constrains the PRD1 regulation domain into an active conformation which is transduced to CAT via a network of negatively charged residues belonging to PRD1 dimeric interface and to the linker region. In addition, our data support a model where BglG-type antitermination regulatory modules can only adopt a single conformation compatible with the active structure of the effector domain, regardless of whether activation is mediated by mutation on the first or second PRD. The linker between the effector and regulation modules appears to function as an adaptable hinge tuning the position of the functional modules.
Assessment of scoring functions to rank the quality of 3D subtomogram clusters from cryo-electron tomography.
Venue: Journal of structural biology

Cryo-electron tomography provides the opportunity for unsupervised discovery of endogenous complexes in situ. This process usually requires particle picking, clustering and alignment of subtomograms to produce an average structure of the complex. When applied to heterogeneous samples, template-free clustering and alignment of subtomograms can potentially lead to the discovery of structures for unknown endogenous complexes. However, such methods require scoring functions to measure and accurately rank the quality of aligned subtomogram clusters, which can be compromised by contaminations from misclassified complexes and alignment errors. Here, we provide the first study to assess the effectiveness of more than 15 scoring functions for evaluating the quality of subtomogram clusters, which differ in the amount of structural misalignments and contaminations due to misclassified complexes. We assessed both experimental and simulated subtomograms as ground truth data sets. Our analysis showed that the robustness of scoring functions varies largely. Most scores were sensitive to the signal-to-noise ratio of subtomograms and often required Gaussian filtering as preprocessing for improved performance. Two scoring functions, Spectral SNR-based Fourier Shell Correlation and Pearson Correlation in the Fourier domain with missing wedge correction, showed a robust ranking of subtomogram clusters without any preprocessing and irrespective of SNR levels of subtomograms. Of these two scoring functions, Spectral SNR-based Fourier Shell Correlation was fastest to compute and is a better choice for handling large numbers of subtomograms. Our results provide a guidance for choosing an accurate scoring function for template-free approaches to detect complexes from heterogeneous samples.
An exon-biased biophysical approach and NMR spectroscopy define the secondary structure of a conserved helical element within the HOTAIR long non-coding RNA.
Venue: Journal of structural biology

HOTAIR is a large, multi-exon spliced non-coding RNA proposed to function as a molecular scaffold and competes with chromatin to bind to histone modification enzymes. Previous sequence analysis and biochemical experiments identified potential conserved regions and characterized the full length HOTAIR secondary structure. Here, we examine the thermodynamic folding properties and structural propensity of the individual exonic regions of HOTAIR using an array of biophysical methods and NMR spectroscopy. We demonstrate that different exons of HOTAIR contain variable degrees of heterogeneity, and identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing unambiguously confirm conserved base pair interactions within the structural element, termed helix 10 of exon 4, located within domain I of human HOTAIR. This combined exon-biased and integrated biophysical approach introduces a new strategy to examine conformational heterogeneity in lncRNAs and emphasizes NMR as a key method to validate base pair interactions and corroborate large RNA secondary structures.
Structure of a consensus chitin-binding domain revealed by solution NMR.
Venue: Journal of structural biology

Chitin-binding proteins (CBPs) are a versatile group of proteins found in almost every organism on earth. CBPs are involved in enzymatic carbohydrate degradation and also serve as templating scaffolds in the exoskeleton of crustaceans and insects. One specific chitin-binding motif found across a wide range of arthropods' exoskeletons is the "extended Rebers and Riddiford" consensus (R&R), whose mechanism of chitin binding remains unclear. Here, we report the 3D structure and molecular level interactions of a chitin-binding domain (CBD-γ) located in a CBP from the beak of the jumbo squid Dosidicus gigas. This CBP is one of four chitin-binding proteins identified in the beak mouthpart of D. gigas and is believed to interact with chitin to form a scaffold network that is infiltrated with a second set of structural proteins during beak maturation. We used solution state NMR spectroscopy to elucidate the molecular interactions between CBD-γ and the soluble chitin derivative pentaacetyl-chitopentaose (PCP) and find that folding of this domain is triggered upon its interaction with PCP. To our knowledge, this is the first experimental 3D structure of a CBP containing the R&R consensus motif, which can be used as a template to understand in more details the role of the R&R motif found in a wide range of CBP-chitin complexes. The present structure also provides molecular information for biomimetic synthesis of graded biomaterials using aqueous-based chemistry and biopolymers.
Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase.
Venue: Journal of structural biology

Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur for biomass incorporation. Electron transfer within SiR requires intra- and inter-subunit interactions that are mediated by the relative position of each protein, governed by flexible domain movements. Using small-angle neutron scattering, we report the first solution structures of SiR heterodimers containing a single copy of each subunit. These structures show how the subunits bind and how both subunit binding and oxidation state impact SiRFP's conformation. Neutron contrast matching experiments on selectively deuterated heterodimers allow us to define the contribution of each subunit to the solution scattering. SiRHP binding induces a change in the position of SiRFP's flavodoxin-like domain relative to its ferredoxin-NADP+ reductase domain while compacting SiRHP's N-terminus. Reduction of SiRFP leads to a more open structure relative to its oxidized state, re-positioning SiRFP's N-terminal flavodoxin-like domain towards the SiRHP binding position. These structures show, for the first time, how both SiRHP binding to, and reduction of, SiRFP positions SiRFP for electron transfer between the subunits.
Rapid tilt-series method for cryo-electron tomography: Characterizing stage behavior during FISE acquisition.
Venue: Journal of structural biology

We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals.
NMR structure and dynamics of Q4DY78, a conserved kinetoplasid-specific protein from Trypanosoma cruzi.
Venue: Journal of structural biology

The 106-residue protein Q4DY78 (UniProt accession number) from Trypanosoma cruzi is highly conserved in the related kinetoplastid pathogens Trypanosoma brucei and Leishmania major. Given the essentiality of its orthologue in T. brucei, the high sequence conservation with other trypanosomatid proteins, and the low sequence similarity with mammalian proteins, Q4DY78 is an attractive protein for structural characterization. Here, we solved the structure of Q4DY78 by solution NMR and evaluated its backbone dynamics. Q4DY78 is composed of five α -helices and a small, two-stranded antiparallel β-sheet. The backbone RMSD is 0.22 ± 0.05 Å for the representative ensemble of the 20 lowest-energy structures. Q4DY78 is overall rigid, except for N-terminal residues (V8 to I10), residues at loop 4 (K57 to G65) and residues at the C-terminus (F89 to F112). Q4DY78 has a short motif FPCAP that could potentially mediate interactions with the host cytoskeleton via interaction with EVH1 (Drosophila Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1) domains. Albeit Q4DY78 lacks calcium-binding motifs, its fold resembles that of eukaryotic calcium-binding proteins such as calcitracin, calmodulin, and polcacin Bet V4. We characterize this novel protein with a calcium binding fold without the capacity to bind calcium.
DeepAlign, a 3D Alignment Method based on Regionalized Deep Learning for Cryo-EM.
Venue: Journal of structural biology

Cryo Electron Microscopy (Cryo-EM) is currently one of the main tools to reveal the structural information of biological specimens at high resolution. Despite the great development of the techniques involved to solve the biological structures with Cryo-EM in the last years, the reconstructed 3D maps can present lower resolution due to errors committed while processing the information acquired by the microscope. One of the main problems comes from the 3D alignment step, which is an error-prone part of the reconstruction workflow due to the very low signal-to-noise ratio (SNR) common in Cryo-EM imaging. In fact, as we will show in this work, it is not unusual to find a disagreement in the alignment parameters in approximately 20-40% of the processed images, when outputs of different alignment algorithms are compared. In this work, we present a novel method to align sets of single particle images in the 3D space, called DeepAlign. Our proposal is based on deep learning networks that have been successfully used in plenty of problems in image classification. Specifically, we propose to design several deep neural networks on a regionalized basis to classify the particle images in sub-regions and, then, make a refinement of the 3D alignment parameters only inside that sub-region. We show that this method results in accurately aligned images, improving the Fourier shell correlation (FSC) resolution obtained with other state-of-the-art methods while decreasing computational time.
Calmodulin binds to the STAS domain of SLC26A5 prestin with a calcium-dependent, one-lobe, binding mode.
Venue: Journal of structural biology

SLC26A5 transporter prestin is fundamental for the higher hearing sensitivity and frequency selectivity of mammals. Prestin is a voltage-dependent transporter found in the cochlear outer hair cells responsible for their electromotility. Intracellular chloride binding is considered essential for voltage sensitivity and electromotility. Prestin is composed by a transmembrane domain and by a cytosolic domain called STAS. There is evidence of a calcium/calmodulin regulation of prestin mediated by the STAS domain. Using different biophysical techniques, namely SEC, CD, ITC, MST, NMR and SAXS here we demonstrate and characterize the direct interaction between calmodulin and prestin STAS. We show that the interaction is calcium-dependent and that involves residues at the N-terminal end of the "variable loop". This is an intrinsically disordered insertion typical of the STAS domains of the SLC26 family of transporters whose function is still unclear. We derive a low-resolution model of the STAS/CaM complex, where only one lobe of calmodulin is engaged in the interaction, and build a model for the entire dimeric prestin in complex with CaM, which can use the unoccupied lobe to interact with other regions of prestin or with other regulatory proteins. We show that also a non-mammalian STAS can interact with calmodulin via the variable loop. These data start to shed light on the regulatory role of the STAS variable loop of prestin.
Spike protein fusion loop controls SARS-CoV-2 fusogenicity and infectivity
Venue: Journal of Structural Biology

The high SARS-CoV-2 reproductive number driving the COVID-19 pandemic has been a mystery. Our recent in vitro, and in vivo coronaviral pathogenesis studies involving Mouse Hepatitis Virus (MHV-A59) suggest a crucial role for a small host membrane-virus contact initiator region of the Spike protein, called the fusion peptide that enhances the virus fusogenicity and infectivity. Here we study the Spike from five human β-coronaviruses (HCoV) including the SARS-CoV-2, and MHV-A59 for comparison. The structural and dynamics analyses of the Spike show that its fusion loop spatially organizes three fusion peptides contiguous to each other to synergistically trigger the virus-host membrane fusion process. We propose a Contact Initiation Model based on the architecture of the Spike quaternary structure that explains the obligatory participation of the fusion loop in the initiation of the host membrane contact for the virus fusion process. Among all the HCoV Spikes in this study, SARS-CoV-2 has the most hydrophobic surface and the extent of hydrophobicity correlates with the reproductive number and infectivity of the other HCoV. Comparison between results from standard and replica exchange molecular dynamics reveal the unique physicochemical properties of the SARS-CoV-2 fusion peptides, accrued in part from the presence of consecutive prolines that impart backbone rigidity which aids the virus fusogenicity. The priming of the Spike by its cleavage and subsequent fusogenic conformational transition steered by the fusion loop may be critical for the SARS-CoV-2 spread. The importance of the fusion loop makes it an apt target for anti-virals and vaccine candidates.
Porous Assembly of an Antifungal Protein Mediated by Zinc and Sulfonato-calix[8]arene.
Venue: Journal of structural biology

Controlled protein assembly holds great potential in the fabrication of biohybrid materials. However, the structural diversity and complexity of proteins present an obstacle to controlled assembly. Supramolecular chemistry is a possible solution as it offers tools to mediate self-assembly with molecular precision. This paper deals with the calixarene- and zinc-mediated assembly and crystallization of the histidine-rich Penicillium chrysogenum antifungal protein B (PAFB). We report crystal structures of pure PAFB, PAFB in complex with Zn2+, and the ternary complex of PAFB, Zn2+ and sulfonato-calix[8]arene (sclx8). A comparison of the three crystal structures revealed the structural plasticity of PAFB. While the flexible and highly anionic sclx8 resulted in large molecular weight aggregates of PAFB in solution, diffraction-quality crystals of PAFB-sclx8 were not obtained. We report crystals of PAFB-Zn2+-sclx8 in which a trinuclear zinc cluster occurred adjacent to a calixarene binding site. Interestingly, the combination of sclx8 complexation and zinc coordination resulted in a porous framework with channels of circa 2 nm diameter. Detailed analysis of the crystal structure highlighted novel molecular recognition features. This research enriches the set of supramolecular interactions available to promote protein assembly.
Anti-NSCLC activity in vitro of Hsp90N inhibitor KW-2478 and complex crystal structure determination of Hsp90N-KW-2478.
Venue: Journal of structural biology

KW-2478 is a promising anti-cancer lead compound targeting to the molecular chaperone heat shock protein 90N (Hsp90N). Absence of complex crystal structure of Hsp90N-KW-2478, however, hampered further structure optimization of KW-2478 and understanding on the molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90N-KW-2478 was determined by X-ray diffraction (XRD, resolution limit: 1.59 Å; PDB ID: 6LT8) and their molecular interaction was analyzed in detail, which suggested that KW-2478 perfectly bound in the N-terminal ATP-binding pocket of Hsp90 to disable its molecular chaperone function, therefore suppressed or killed cancer cells. The results from thermal shift assay (TSA, ΔTm, 18.82±0.51°C) and isothermal titration calorimetry (ITC, Kd, 7.30±2.20 nM) suggested that there is an intense binding force and favorable thermodynamic changes during the process of KW-2478 binding with Hsp90N. Additionally, KW-2478 exhibited favorable anti-NSCLC activity in vitro, as it inhibited cell proliferation (IC50, 8.16 μM for A549; 14.29 μM for H1975) and migration, induced cell cycle arrest and promoted apoptosis. Thirty-six novel KW-2478 derivatives were designed, based on the complex crystal structure and molecular interaction analysis of Hsp90N-KW-2478 complex. Among them, twenty-two derivatives exhibited increased binding force with Hsp90N evaluated by molecular docking assay. The results would provide new guidance for anti-NSCLC new drug development based on the lead compound KW-2478.
CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy.
Venue: Journal of structural biology

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support automated cryo-EM data acquisition. Here, we present CorRelator: an open-source desktop application that bridges between cryo-FLM and real-time cryo-EM/ET automated data collection. CorRelator implements a pixel-coordinate-to-stage-position transformation for flexible, high accuracy on-the-fly and post-acquisition correlation. CorRelator can be integrated into cryo-CLEM workflows and easily adapted to standard fluorescence and transmission electron microscope (TEM) system configurations. CorRelator was benchmarked under live-cell and cryogenic conditions using several FLM and TEM instruments, demonstrating that CorRelator reliably supports real-time, automated correlative cryo-EM/ET acquisition, through a combination of software-aided and interactive alignment. CorRelator is a cross-platform software package featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at:https://github.com/wright-cemrc-projects/corr.
3D Variability Analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM.
Venue: Journal of structural biology

Single particle cryo-EM excels in determining static structures of protein molecules, but existing 3D reconstruction methods have been ineffective in modelling flexible proteins. We introduce 3D variability analysis (3DVA), an algorithm that fits a linear subspace model of conformational change to cryo-EM data at high resolution. 3DVA enables the resolution and visualization of detailed molecular motions of both large and small proteins, revealing new biological insight from single particle cryo-EM data. Experimental results demonstrate the ability of 3DVA to resolve multiple flexible motions of α-helices in the sub-50 kDa transmembrane domain of a GPCR complex, bending modes of a sodium ion channel, five types of symmetric and symmetry-breaking flexibility in a proteasome, large motions in a spliceosome complex, and discrete conformational states of a ribosome assembly. 3DVA is implemented in the cryoSPARC software package.
Calcite crystal orientation patterns in the bilayers of laminated shells of benthic rotaliid foraminifera.
Venue: Journal of structural biology

Shells of calcifying foraminifera play a major role in marine biogeochemical cycles; fossil shells form important archives for paleoenvironment reconstruction. Despite their importance in many Earth science disciplines, there is still little consensus on foraminiferal shell mineralization. Geochemical, biochemical, and physiological studies showed that foraminiferal shell formation might take place through various and diverse mineralization mechanisms. In this study, we contribute to benthic foraminiferal shell calcification through deciphering crystallite organization within the shells. We base our conclusions on results gained from electron backscattered diffraction (EBSD) measurements and describe microstructure/texture characteristics within the laminated shell walls of the benthic, symbiontic foraminifera: Ammonia tepida, Amphistegina lobifera, Amphistegina lessonii. We highlight crystallite assembly patterns obtained on differently oriented cuts and discuss crystallite sizes, morphologies, interlinkages, orientations, co-orientation strengths. We show that: (i) crystals within benthic foraminiferal shells are mesocrystals, (ii) have dendritic-fractal morphologies and (iii) interdigitate strongly. Based on crystal size, we (iv) differentiate between the two layers that comprise the shells and demonstrate that (v) crystals in the septa have different assemblies relative to those in the shell walls. We highlight that (vi) at junctions of different shell elements the axis of crystal orientation jumps abruptly such that their assembly in EBSD maps has a bimodal distribution. We proof (vii) extensive twin-formation within foraminiferal calcite and demonstrate (viii) the presence of two twin modes: 60°/[001] and 77°/∼[6-61] and visualize their distributions within the shells. In a broader perspective, we draw conclusions on processes that lead to the observed microstructure/texture patterns.