nils-herrmann/clean_scirepeval huggingface dataset:
Structural characterization of proteins and complexes using small-angle X-ray solution scattering.
Venue: Journal of structural biology

Small-angle scattering of X-rays (SAXS) is an established method for the low-resolution structural characterization of biological macromolecules in solution. The technique provides three-dimensional low-resolution structures, using ab initio and rigid body modeling, and allow one to assess the oligomeric state of proteins and protein complexes. In addition, SAXS is a powerful tool for structure validation and the quantitative analysis of flexible systems, and is highly complementary to the high resolution methods of X-ray crystallography and NMR. At present, SAXS analysis methods have reached an advanced state, allowing for automated and rapid characterization of protein solutions in terms of low-resolution models, quaternary structure and oligomeric composition. In this communication, main approaches to the characterization of proteins and protein complexes using SAXS are reviewed. The tools for the analysis of proteins in solution are presented, and the impact that these tools have made in modern structural biology is discussed.
GPU-enabled FREALIGN: accelerating single particle 3D reconstruction and refinement in Fourier space on graphics processors.
Venue: Journal of structural biology

Among all the factors that determine the resolution of a 3D reconstruction by single particle electron cryo-microscopy (cryoEM), the number of particle images used in the dataset plays a major role. More images generally yield better resolution, assuming the imaged protein complex is conformationally and compositionally homogeneous. To facilitate processing of very large datasets, we modified the computer program, FREALIGN, to execute the computationally most intensive procedures on Graphics Processing Units (GPUs). Using the modified program, the execution speed increased between 10 and 240-fold depending on the task performed by FREALIGN. Here we report the steps necessary to parallelize critical FREALIGN subroutines and evaluate its performance on computers with multiple GPUs.
Mechanism of substrate specificity in 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases.
Venue: Journal of structural biology

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5'-alkylthio binding site in Arabidopsis thalianaAtMTAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein-SAH complex at 2.2Å resolution. The lack of catalytic activity appears to be related to the enzyme's inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium-hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN.
Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata.
Venue: Journal of structural biology

An integrated study of shell formation was initiated covering the entire life cycle of the marine gastropod Haliotis tuberculata. Shell microstructure, chemistry and mineralogy were investigated by polarized microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and infra-red (IR) spectroscopy. SEM images of trochophore and veliger larvae showed the different stages of shell growth from the initial shell field to the late calcified protoconch. Cross-sections revealed the microstructural arrangement of biominerals, showing the progressive mineralization of the organic protoconch prior to metamorphosis. To gain more information on mineralogical composition, EDX analyses and IR spectroscopy were performed along the development stages. The results demonstrated that early protoconch was mostly composed of amorphous calcium carbonate, while veliger stages showed a gradually crystallization under the form of aragonite. Post-metamorphic shell contained two distinct parts, the original protoconch supporting the new juvenile shell characterized by a marked sculptural pattern. The shells from post-larval and juvenile abalones were essentially made of aragonite.
Structure of the novel 14kDa fragment of alpha-subunit of phycoerythrin from the starving cyanobacterium Phormidium tenue.
Venue: Journal of structural biology

The rod-like phycobilisome (PBS) in cyanobacterium is the light-harvesting complex of phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). The orderly degradation of PBS was observed under starvation conditions. A 14 kDa truncated fragment of alpha-subunit of PE (F-alphaPE) was identified from the degraded product. F-alphaPE was purified to homogeneity, sequenced and crystallized. The merohedrally twinned crystals with a twinning factor of approximately 0.5 were obtained. The crystal structure of F-alphaPE was determined with molecular replacement method using detwinned data and refined to an R(cryst) factor of 23.2% (R(free)=27.6%). The structure consisted of two crystallographically independent molecules in the asymmetric unit. The two molecules were designated as molecules A and B with a buried area of 200 A(2) at the interface. The structure of F-alphaPE consists of seven alpha-helices A, B, E, F, F', G and H. The first 31N-terminal residues that fold into parallel alpha-helices X and Y in other PEs are not present in the amino acid sequence of F-alphaPE. Both molecules, A and B contain two chromophore ligands, PEB1 and PEB2 in each. These are covalently linked to the polypeptide chain through Cys82 and Cys139, respectively. The superimposition of C(alpha) tracings of molecules A and B shows an r.m.s. shift of 1.0 A indicating that the structures of two independent molecules are very similar. The degradation of phycobilisome proteins under starvation stress seems to occur to supplement the requirement of amino acids for protein synthesis and to reduce the absorption of light energy.
Crystal structures of a novel anti-HIV mannose-binding lectin from Polygonatum cyrtonema Hua with unique ligand-binding property and super-structure.
Venue: Journal of structural biology

Polygonatum cyrtonema lectin (PCL) is a novel anti-HIV mannose-binding lectin from Galanthus nivalis agglutinin (GNA)-related lectin family. Crystal structures of ligand-free PCL and its complexes with monomannoside and alpha1-3 dimannoside have been determined. The ligand-free PCL is dimeric, with both subunits adopt the beta-prism II fold. PCL subunit binds mannose using a potential bivalent mode instead of the usual trivalent mode, in which carbohydrate-binding site (CBS) I and CBS III adopt the conserved mannose-binding motif of QXDXNXVXY (X is one of any amino acid residues) as observed in other structurally characterized GNA-related lectins, while CBS II adopts a modified motif with residues Gln58 and Asp60, which are critical for mannose-binding, substituted by His58 and Asn60, respectively. As a result, CBS II is unfit for mannose-binding. In the mannoside complexes, ligand-bindings only occur at CBS I which provides the specificity for alpha1-3 dimannoside. CBS II and CBS III are cooperatively occupied by a well-ordered sulfate ion, through which the individual dimers are cross-linked to form a unique super-structure of 3(2) helical lattice. Surveying the sequences of GNA-related lectins revealed that the modified binding motif of CBS II is widely distributed in the Liliaceae family as an intrinsic structural element. There is evidence that other GNA-related lectins will also adopt the similar super-structure as PCL. Thus PCL structure, unique in ligand-binding mode, may represent a novel type of structure of GNA-related lectins. Comparative analyses indicated that the dimer-based super-structure may play a primary role in the anti-HIV property of PCL.
High-throughput production of human proteins for crystallization: The SGC experience
Venue: Journal of structural biology

Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of ∼40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9–20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.
X-ray microCT imaging technique reveals corm microstructures of an arctic-boreal cotton-sedge, Eriophorum vaginatum.
Venue: Journal of structural biology

X-ray computed tomography (CT), a non-destructive imaging technique, has recently been effectively applied to botanical research. In this study an X-ray microCT technique was developed to allow for anatomical study of the overwintering corms of Eriophorum vaginatum, an ecologically important sedge species in arctic tussock-tundra and boreal peatlands. Using a GE Medical MS8X-130 X-ray microCT scanner, optimal imaging parameters included scanning isolated corms at 80 k Vp and 100 microA with a 3500 ms exposure time and an isotropic voxel size of 10 microm. A Gaussian blur image filter with a blur radius (sigma) of two pixels was applied to the optimal dataset to improve visual detection and contrast of tissues while removing 99.2% of image noise. Using the developed X-ray microCT technique several undocumented anatomical characteristics of the corm were identified including the vascular connection between a parent corm and branching cormel and the 3D shape of sclereid clusters. The 3D structure of sclereid clusters was determined whereby the perimeter of their lance shape is greatly reinforced by sclereids with thicker secondary cell walls as compared to those of the interior of the cluster. The structure of sclereid clusters and their association with leaf traces suggests they may be stabilizing the corm-leaf connection to protect vascular tissues from physical damage. The proposed X-ray microCT technique is an excellent tool for determination of the 3D structure of E. vaginatum corms and may be used to detect alterations in tissue structure and chemistry in response to environmental change in this and other Cyperaceous species.
Solution structure and phospho-PmrA recognition mode of PmrD from Klebsiella pneumoniae.
Venue: Journal of structural biology

In bacteria, the two-component system (TCS) is the most prevalent for sensing and transducing the environmental signals into the cell. In Salmonella, the small basic protein PmrD is found to protect phospho-PmrA and prolong the expression of PmrA-activated genes. In contrast, Escherichia coli PmrD fails to protect phospho-PmrA. Here, we show that Klebsiella pneumoniae PmrD (KP-PmrD) can inhibit the dephosphrylation of phospho-PmrA, and the interaction between KP-PmrD and the N-terminal receiver domain of PmrA (PmrA(N)) is much stronger in the presence than in the absence of the phosphoryl analog beryllofluoride (BeF(3)(-)) (K(D)=1.74 ± 0.81 μM vs. K(D)=236 ± 48 μM). To better understand the molecular interactions involved, the solution structure of KP-PmrD was found to comprise six β-strands and a flexible C-terminal α-helix. Amide chemical shift perturbations of KP-PmrD in complex with BeF(3)(-)-activated PmrA(N) suggested that KP-PmrD may undergo a certain conformational rearrangement on binding to activated PmrA(N). Saturation transfer experiments revealed the binding surface to be located on one face of the β-barrel. This finding was further verified by in vivo polymyxin B susceptibility assay of the mutants of KP-PmrD. The phospho-PmrA recognition surface of KP-PmrD, which involves two KP-PmrD proteins in complex with an activated-PmrA(N) dimer, is suggested to be a contiguous patch consisting of Trp3, Trp4, Ser23, Leu26, Glu27, Met28, Thr46, Leu48, Ala49, Asp50, Ala51, Arg52, Ile65, Asn67, Ala68, Thr69, His70, Tyr71, Ser73 and Glu74. Our study furthers the understanding of how PmrD protects phopho-PmrA in the PmrAB TCS.
Cryo-electron tomography of bacteriophage phi6 procapsids shows random occupancy of the binding sites for RNA polymerase and packaging NTPase.
Venue: Journal of structural biology

Assembly of dsRNA bacteriophage phi6 involves packaging of the three mRNA strands of the segmented genome into the procapsid, an icosahedrally symmetric particle with recessed vertices. The hexameric packaging NTPase (P4) overlies these vertices, and the monomeric RNA-dependent RNA polymerase (RdRP, P2) binds at sites inside the shell. P2 and P4 are present in substoichiometric amounts, raising the questions of whether they are recruited to the nascent procapsid in defined amounts and at specific locations, and whether they may co-localize to form RNA-processing assembly lines at one or more "special" vertices. We have used cryo-electron tomography to map both molecules on individual procapsids. The results show variable complements that accord with binomial distributions with means of 8 (P2) and 5 (P4), suggesting that they are randomly incorporated in copy numbers that simply reflect availability, i.e. their rates of synthesis. Analysis of the occupancy of potential binding sites (20 for P2; 12 for P4) shows no tendency to cluster nor for P2 and P4 to co-localize, suggesting that the binding sites for both proteins are occupied in random fashion. These observations indicate that although P2 and P4 act sequentially on the same substrates there is no direct physical coupling between their activities.
An adaptive Expectation-Maximization algorithm with GPU implementation for electron cryomicroscopy.
Venue: Journal of structural biology

Maximum-likelihood (ML) estimation has very desirable properties for reconstructing 3D volumes from noisy cryo-EM images of single macromolecular particles. Current implementations of ML estimation make use of the Expectation-Maximization (EM) algorithm or its variants. However, the EM algorithm is notoriously computation-intensive, as it involves integrals over all orientations and positions for each particle image. We present a strategy to speedup the EM algorithm using domain reduction. Domain reduction uses a coarse grid to evaluate regions in the integration domain that contribute most to the integral. The integral is evaluated with a fine grid in these regions. In the simulations reported in this paper, domain reduction gives speedups which exceed a factor of 10 in early iterations and which exceed a factor of 60 in terminal iterations.
Crystal structure of a fructokinase homolog from Halothermothrix orenii.
Venue: Journal of structural biology

Fructokinase (FRK; EC 2.7.1.4) catalyzes the phosphorylation of d-fructose to d-fructose 6-phosphate (F6P). This irreversible and near rate-limiting step is a central and regulatory process in plants and bacteria, which channels fructose into a metabolically active state for glycolysis. Towards understanding the mechanism of FRK, here we report the crystal structure of a FRK homolog from a thermohalophilic bacterium Halothermothrixorenii (Hore_18220 in sequence databases). The structure of the Hore_18220 protein reveals a catalytic domain with a Rossmann-like fold and a beta-sheet "lid" for dimerization. Based on comparison of Hore_18220 to structures of related proteins, we propose its mechanism of action, in which the lid serves to regulate access to the substrate binding sites. Close relationship of Hore_18220 and plant FRK enzymes allows us to propose a model for the structure and function of FRKs.
Low cost, high performance GPU computing solution for atomic resolution cryoEM single-particle reconstruction.
Venue: Journal of structural biology

Recent advancements in cryo-electron microscopy (cryoEM) have made it technically possible to determine the three-dimensional (3D) structures of macromolecular complexes at atomic resolution. However, processing the large amount of data needed for atomic resolution reconstructions requires either accessing to very expensive computer clusters or waiting for weeks of continuous computation in a personal computer (PC). In this paper, we present a practical computational solution to this 3D reconstruction problem through the optimal utilization of the processing capabilities of both commodity graphics hardware (i.e., general purpose graphics processing unit (GPGPU)). Our solution, which is implemented in a new program, called eLite3D, has a number of advanced features of general interests. First, we construct interleaved schemes to prevent the data race condition intrinsic in merging of 2D data into a 3D volume. Second, we introduce a processing pipeline strategy to optimally balance I/O and computation operations, thus improving CPU and GPGPU parallelism. The speedup of eLite3D is up to 100 times over other commonly used 3D reconstruction programs with the same accuracy, thus allowing completion of atomic resolution 3D reconstructions of large complexes in a PC in 1-2h other than days or weeks. Our result provides a practical solution to atomic resolution cryoEM (asymmetric or symmetric) reconstruction and offers useful guidelines for developing GPGPU applications in general.
Biophysical characterization of recombinant proteins: A key to higher structural genomics success
Venue: Journal of structural biology

Hundreds of genomes have been successfully sequenced to date, and the data are publicly available. At the same time, the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts. Frequently, however, little is known about newly expressed proteins calling for large-scale protein characterization to better understand their biochemical roles and to enable structure–function relationship studies. In the Structural Genomics Consortium (SGC), we have established a platform to characterize large numbers of purified proteins. This includes screening for ligands, enzyme assays, peptide arrays and peptide displacement in a 384-well format. In this review, we describe this platform in more detail and report on how our approach significantly increases the success rate for structure determination. Coupled with high-resolution X-ray crystallography and structure-guided methods, this platform can also be used toward the development of chemical probes through screening families of proteins against a variety of chemical series and focused chemical libraries.
Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor.
Venue: Journal of structural biology

G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.
FtsH cleavage of non-native conformations of proteins.
Venue: Journal of structural biology

FtsH is a peculiar prokaryotic protease with low unfoldase activity. Different reports have proposed that FtsH substrates could be either tagged proteins or proteins of low stability. We show here that FtsH degradation of 31 point mutants of Anabaena apoflavodoxin is inversely proportional to their conformational stabilities, and that the same applies to other substrate proteins. In contrast, highly stable proteins such as GST and holoflavodoxin are not degraded at all. Attempts to identify sequence tags signaling for degradation in apoflavodoxin fragments have been unsuccessful. Apoflavodoxin adopts three conformations: native, partly unfolded and fully unfolded. It is revealing that degradation of the 31 variants is proportional to the molar fraction of fully unfolded molecules and inversely proportional to the fraction of stable apoflavodoxin molecules. This indicates that FtsH, rather than unfolding the protein, acts on the fraction that is already unfolded.
Visualization of inositol 1,4,5-trisphosphate receptors on the nuclear envelope outer membrane by freeze-drying and rotary shadowing for electron microscopy.
Venue: Journal of structural biology

The receptors for the second messenger InsP(3) comprise a family of closely related ion channels that release Ca(2+) from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP(3)Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca(2+) signals, but the spatial organization of InsP(3)Rs in Ca(2+) stores is poorly characterized. Using nuclei isolated from insect Sf9 cells and freeze-dry rotary shadowing, we have addressed this by directly visualizing the cytoplasmic domain of InsP(3)R located on the cytoplasmic side of the nuclear envelope. Identification of approximately 15 nm structures as the cytoplasmic domain of InsP(3)R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP(3)R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP(3)R antibody. Over-expression of InsP(3)R did not result in the formation of arrays or clusters with channels touching each other. Gold-labeling suggests that the channel amino terminus resides near the center of the cytoplasmic tetrameric quaternary structure. The combination of nuclear isolation with freeze-drying and rotary shadow techniques allows direct visualization of InsP(3)Rs in native nuclear envelopes and can be used to determine their spatial distribution and density.