nils-herrmann/clean_scirepeval huggingface dataset:
Crystal structure of the pilotin from the enterohemorrhagic Escherichia coli type II secretion system.
Venue: Journal of structural biology

Bacteria contain several sophisticated macromolecular machineries responsible for translocating proteins across the cell envelope. One prominent example is the type II secretion system (T2SS), which contains a large outer membrane channel, called the secretin. These gated channels require specialized proteins, so-called pilotins, to reach and assemble in the outer membrane. Here we report the crystal structure of the pilotin GspS from the T2SS of enterohemorrhagic Escherichia coli (EHEC), an important pathogen that can cause severe disease in cases of food poisoning. In this four-helix protein, the straight helix α2, the curved helix α3 and the bent helix α4 surround the central N-terminal helix α1. The helices of GspS create a prominent groove, mainly formed by side chains of helices α1, α2 and α3. In the EHEC GspS structure this groove is occupied by extra electron density which is reminiscent of an α-helix and corresponds well with a binding site observed in a homologous pilotin. The residues forming the groove are well conserved among homologs, pointing to a key role of this groove in this class of T2SS pilotins. At the same time, T2SS pilotins in different species can be entirely different in structure, and the pilotins for secretins in non-T2SS machineries have yet again unrelated folds, despite a common function. It is striking that a common complex function, such as targeting and assembling an outer membrane multimeric channel, can be performed by proteins with entirely different folds.
Automatic post-picking using MAPPOS improves particle image detection from Cryo-EM micrographs
Venue: Journal of structural biology

Cryo-electron microscopy (cryo-EM) studies using single particle reconstruction are extensively used to reveal structural information on macromolecular complexes. Aiming at the highest achievable resolution, state of the art electron microscopes automatically acquire thousands of high-quality micrographs. Particles are detected on and boxed out from each micrograph using fully- or semi-automated approaches. However, the obtained particles still require laborious manual post-picking classification, which is one major bottleneck for single particle analysis of large datasets. We introduce MAPPOS, a supervised post-picking strategy for the classification of boxed particle images, as additional strategy adding to the already efficient automated particle picking routines. MAPPOS employs machine learning techniques to train a robust classifier from a small number of characteristic image features. In order to accurately quantify the performance of MAPPOS we used simulated particle and non-particle images. In addition, we verified our method by applying it to an experimental cryo-EM dataset and comparing the results to the manual classification of the same dataset. Comparisons between MAPPOS and manual post-picking classification by several human experts demonstrated that merely a few hundred sample images are sufficient for MAPPOS to classify an entire dataset with a human-like performance. MAPPOS was shown to greatly accelerate the throughput of large datasets by reducing the manual workload by orders of magnitude while maintaining a reliable identification of non-particle images.
Crystal structure analysis of human Sirt2 and its ADP-ribose complex.
Venue: Journal of structural biology

Sirtuins are NAD(+)-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.
Marker-free dual-axis tilt series alignment.
Venue: Journal of structural biology

Dual-axis tilt series in electron tomography are collected by successively tilting the object about two approximately orthogonal tilt axes. Here we report on the extension of marker-free image registration based on cross-correlation techniques to dual-axis tilt series. A simultaneous geometry refinement yields accurate parameters for the computation of the final reconstruction. Both, image registration and 3D-reconstruction operate on the combined data from the paired single axis series rather than computing individual single axis tomograms followed by a separate combination step. We show that with simultaneous registration and reconstruction of dual-axis tilt series, tomograms with higher resolution are obtained than by merging separately computed tomograms.
Maskiton: Interactive, web-based classification of single-particle electron microscopy images.
Venue: Journal of structural biology

Electron microscopy (EM) is an important tool for determining the composition, arrangement and structure of biological macromolecules. When studying structurally heterogeneous samples using EM, classification is a critical step toward achieving higher resolution and identifying biologically significant conformations. We have developed an interactive, web-based tool, called Maskiton, for creating custom masks and performing 2D classifications on aligned single-particle EM images. The Maskiton interface makes it considerably easier and faster to explore the significance of heterogeneity in single-particle datasets. Maskiton features include: resumable uploads to facilitate transfer of large datasets to the server, custom mask creation in the browser, continual progress updates, and interactive viewing of classification results. To demonstrate the value of this tool, we provide examples of its use on several experimental datasets and include analyses of the independent terminus mobility within the Ltn1 E3 ubiquitin ligase, the in vitro assembly of 30S ribosomal subunits, and classification complexity reduction within Immunoglobulin M. This work also serves as a proof-of-concept for the development of future cross-platform, interactive user interfaces for electron microscopy data processing.
Fourier-space TEM reconstructions with symmetry adapted functions for all rotational point groups.
Venue: Journal of structural biology

A general-purpose and simple expression for the coefficients of symmetry adapted functions referred to conveniently oriented symmetry axes is given for all rotational point groups. The expression involves the computation of reduced Wigner-matrix elements corresponding to an angle specific to each group and has the computational advantage of leading to Fourier-space TEM (transmission electron microscopy) reconstruction procedures involving only real valued unknowns. Using this expression, a protocol for ab initio view and center assignment and reconstruction so far used for icosahedral particles has been tested with experimental data in other point groups.
Consensus among multiple approaches as a reliability measure for flexible fitting into cryo-EM data.
Venue: Journal of structural biology

Cryo-electron microscopy (cryo-EM) can provide low-resolution density maps of large macromolecular assemblies. As the number of structures deposited in the Protein Data Bank by fitting a high-resolution structure into a low-resolution cryo-EM map is increasing, there is a need to revise the protocols and improve the measures for fitting. A recent study suggested using a combination of multiple automated flexible fitting approaches to improve the interpretation of cryo-EM data. The current work further explores the use of multiple approaches by validating this "consensus" fitting approach and deriving a local reliability measure. Here four different flexible fitting approaches are applied for fitting an initial structure into a simulated density map of known target structure from a dataset of proteins. It is found that the models produced from different approaches often have a consensus in conformation and are also near to the target structure, whereas cases not showing consensus are away from the target. A high correlation is also observed between the RMSF profiles calculated with respect to the average and the target structures, which indicates that the relation between consensus and accuracy can also be extended to a per-residue level. Therefore, the RMSF among the fitted models is proposed as a local reliability measure, which can be used to assess the reliability of the fit at specific regions. Hence, we encourage the community to use consensus flexible fitting with different methods to report on local reliability of the resulting models and improve the interpretation of cryo-EM data.
Crystal structure of phospholipase A1 from Streptomyces albidoflavus NA297.
Venue: Journal of structural biology

The metal-independent lipase from Streptomyces albidoflavus NA297 (SaPLA1) is a phospholipase A1 as it preferentially hydrolyzes the sn-1 acyl ester in glycerophospholipids, yielding a fatty acid and 2-acyl-lysophospholipid. The molecular mechanism underlying the substrate binding by SaPLA1 is currently unknown. In this study, the crystal structure of SaPLA1 was determined at 1.75Å resolutions by molecular replacement. A structural similarity search indicated the highest structural similarity to an esterase from Streptomyces scabies, followed by GDSL family enzymes. The SaPLA1 active site is composed of a Ser-His dyad (Ser11 and His218), whereby stabilization of the imidazole is provided by the main-chain carbonyl oxygen of Ser216, a common variation of the catalytic triad in many serine hydrolases, where this carbonyl maintains the orientation of the active site histidine residue. The hydrophobic pocket and cleft for lipid binding are adjacent to the active site, and are approximately 13-15Å deep and 14-16Å long. A partial polyethylene glycol structure was found in this hydrophobic pocket.
Projection-based volume alignment.
Venue: Journal of structural biology

When heterogeneous samples of macromolecular assemblies are being examined by 3D electron microscopy (3DEM), often multiple reconstructions are obtained. For example, subtomograms of individual particles can be acquired from tomography, or volumes of multiple 2D classes can be obtained by random conical tilt reconstruction. Of these, similar volumes can be averaged to achieve higher resolution. Volume alignment is an essential step before 3D classification and averaging. Here we present a projection-based volume alignment (PBVA) algorithm. We select a set of projections to represent the reference volume and align them to a second volume. Projection alignment is achieved by maximizing the cross-correlation function with respect to rotation and translation parameters. If data are missing, the cross-correlation functions are normalized accordingly. Accurate alignments are obtained by averaging and quadratic interpolation of the cross-correlation maximum. Comparisons of the computation time between PBVA and traditional 3D cross-correlation methods demonstrate that PBVA outperforms the traditional methods. Performance tests were carried out with different signal-to-noise ratios using modeled noise and with different percentages of missing data using a cryo-EM dataset. All tests show that the algorithm is robust and highly accurate. PBVA was applied to align the reconstructions of a subcomplex of the NADH: ubiquinone oxidoreductase (Complex I) from the yeast Yarrowia lipolytica, followed by classification and averaging.
The architecture of the joint head cuticle and its transition to the arthrodial membrane in the terrestrial crustacean Porcellio scaber.
Venue: Journal of structural biology

The cuticle of terrestrial isopods is an interesting model for the study of structure-function relationships in biological composite materials. Its organic matrix has a hierarchically organised structure, and type and phase of the mineral compound can vary. The cuticle forms functionally diverse skeletal elements whose properties are adapted to their specific functions. In order to better understand the relation between structure, composition and function of isopod cuticle, we studied the structure and composition of the joint head that is part of the pereiopod's basis. It consists of a central region, whose shape fits well into the joint socket, and an edge region that is connected to the soft arthrodial membrane and protects the central region from mechanical load. The cuticle architecture of the joint head has local variations in structure and composition. In the central region the cuticle is similar to the previously published tergite cuticle. High concentrations of amorphous calcium phosphate are located in the endocuticle suggesting a coexistence with amorphous calcium carbonate. The edge region has an unexpected organisation characterised by thickening of the epi- and exocuticle and an unusual unidirectional orientation of chitin-protein fibrils within the endocuticle. The concentrations of phosphate are considerably higher than in the central region. The overall differentiation in the cuticular architecture of the edge in comparison to the central region reflects the adaptation to mechanical strains the cuticle has to sustain during contraction of extensor muscles, and to the structural and compositional transition from the edge to the connecting arthrodial membrane.
Lensless coherent imaging of proteins and supramolecular assemblies: Efficient phase retrieval by the charge flipping algorithm.
Venue: Journal of structural biology

Diffractive imaging using the intense and coherent beam of X-ray free-electron lasers opens new perspectives for structural studies of single nanoparticles and biomolecules. Simulations were carried out to generate 3D oversampled diffraction patterns of non-crystalline biological samples, ranging from peptides and proteins to megadalton complex assemblies, and to recover their molecular structure from nanometer to near-atomic resolutions. Using these simulated data, we show here that iterative reconstruction methods based on standard and variant forms of the charge flipping algorithm, can efficiently solve the phase retrieval problem and extract a unique and reliable molecular structure. Contrary to the case of conventional algorithms, where the estimation and the use of a compact support is imposed, our approach does not require any prior information about the molecular assembly, and is amenable to a wide range of biological assemblies. Importantly, the robustness of this ab initio approach is illustrated by the fact that it tolerates experimental noise and incompleteness of the intensity data at the center of the speckle pattern.
From colloidal nanoparticles to a single crystal: new insights into the formation of nacre's aragonite tablets.
Venue: Journal of structural biology

Nacre has long served as a model for understanding the biomineralization mechanism and designing bio-inspired materials. However, its basic building blocks, the aragonite tablets, are still under debate in terms of their fine structure at the nanoscale and corresponding formation mechanism. Here, using a field emission scanning electron microscope (SEM), high resolution transmission electron microscope (HRTEM), and X-ray diffractometer, we comparatively investigate the immature and mature tablet from the green mussel's nacre. We find that: (1) the early immature tablet consists of closely-packed colloidal nanoparticles, which contain nanocrystals surrounded by the amorphous calcium carbonate (ACC) phase. Moreover, these nanocrystals are generally different in shape, size, and orientation; (2) the immature tablet can grow via oriented attachment besides via transformation of the ACC phase; and (3) with growth, the colloidal nanoparticles gradually increase in crystallinity and size until fully crystallized and fused together, leading to a mature tablet that is a monolithic single crystal of aragonite. Based on these findings, we propose a new model showing how the mature tablet evolves from the primary colloidal ACC nanoparticles. We expect this work will provide new insights into the formation of single crystal biominerals via the amorphous precursor route.
The backbone model of the Arabis mosaic virus reveals new insights into functional domains of Nepovirus capsid.
Venue: Journal of structural biology

Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.
X-ray crystallographic and molecular docking studies on a unique chloromuconolactone dehalogenase from Rhodococcus opacus 1CP.
Venue: Journal of structural biology

5-Chloromuconolactone dehalogenase (5-CMLD) is a unique enzyme that catalyzes the conversion of 5-chloromuconolactone into cis-dienelactone in the new modified ortho-pathway of the 3-chlorocatechol degradation by Rhodococcus opacus 1CP. In all other known chlorocatechol pathways the dehalogenation is a spontaneous secondary reaction of the unstable chloromuconate intermediate following the lactonization process catalyzed by the muconate cycloisomerases. The crystallographic structure of the decameric 5-CMLD was solved by Molecular Replacement, using the coordinates of the low resolution structure of the highly homologous muconolactone isomerase, an enzyme of the conventional ortho-pathway. Muconolactone isomerase catalyzes the endocyclic rearrangement of the double bond within the lactone ring of muconolactone to yield 3-oxoadipate enol lactone. Although both 5-CMLD and muconolactone isomerase share the ability to dechlorinate 5-chloromuconolactone, 5-CMLD shows a significant degree of specialization, having lost the capacity to convert its original substrate muconolactone. The active site of 5-CMLD was previously hypothesized to reside in a deep pocket at the interface of two different subunits, on the basis of a muconolactone isomerase structure analysis. In this study we also performed molecular docking calculations that confirmed these previous findings, and allowed us furthermore to determine the residues involved in the catalytic process.
Atomic modeling of cryo-electron microscopy reconstructions--joint refinement of model and imaging parameters.
Venue: Journal of structural biology

When refining the fit of component atomic structures into electron microscopic reconstructions, use of a resolution-dependent atomic density function makes it possible to jointly optimize the atomic model and imaging parameters of the microscope. Atomic density is calculated by one-dimensional Fourier transform of atomic form factors convoluted with a microscope envelope correction and a low-pass filter, allowing refinement of imaging parameters such as resolution, by optimizing the agreement of calculated and experimental maps. A similar approach allows refinement of atomic displacement parameters, providing indications of molecular flexibility even at low resolution. A modest improvement in atomic coordinates is possible following optimization of these additional parameters. Methods have been implemented in a Python program that can be used in stand-alone mode for rigid-group refinement, or embedded in other optimizers for flexible refinement with stereochemical restraints. The approach is demonstrated with refinements of virus and chaperonin structures at resolutions of 9 through 4.5 Å, representing regimes where rigid-group and fully flexible parameterizations are appropriate. Through comparisons to known crystal structures, flexible fitting by RSRef is shown to be an improvement relative to other methods and to generate models with all-atom rms accuracies of 1.5-2.5 Å at resolutions of 4.5-6 Å.
A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy
Venue: Journal of structural biology

Difficulties associated with using X-ray crystallography for structural studies of large macromolecular complexes have made single particle cryo-electron microscopy (cryoEM) a key technique in structural biology. The efficient application of the single particle cryoEM approach requires the sample to be vitrified within the holes of carbon films, with particles well dispersed throughout the ice and adopting multiple orientations. To achieve this, the carbon support film is first hydrophilised by glow discharge, which allows the sample to spread over the film. Unfortunately, for transmembrane complexes especially, this procedure can result in severe sample adsorption to the carbon support film, reducing the number of particles dispersed in the ice. This problem is rate-limiting in the single particle cryoEM approach and has hindered its widespread application to hydrophobic complexes. We describe a novel grid preparation technique that allows for good particle dispersion in the ice and minimal hydrophobic particle adhesion to the support film. This is achieved by hydrophilisation of the carbon support film by the use of selected detergents that interact with the support so as to achieve a hydrophilic and neutral or selectively charged surface.