nils-herrmann/clean_scirepeval huggingface dataset:
Constructing atomic structural models into cryo-EM densities using molecular dynamics - Pros and cons.
Venue: Journal of structural biology

Accurate structure determination from electron density maps at 3-5 Å resolution necessitates a balance between extensive global and local sampling of atomistic models, yet with the stereochemical correctness of backbone and sidechain geometries. Molecular Dynamics Flexible Fitting (MDFF), particularly through a resolution-exchange scheme, ReMDFF, provides a robust way of achieving this balance for hybrid structure determination. Employing two high-resolution density maps, namely that of β-galactosidase at 3.2 Å and TRPV1 at 3.4 Å, we showcase the quality of ReMDFF-generated models, comparing them against ones submitted by independent research groups for the 2015-2016 Cryo-EM Model Challenge. This comparison offers a clear evaluation of ReMDFF's strengths and shortcomings, and those of data-guided real-space refinements in general. ReMDFF results scored highly on the various metric for judging the quality-of-fit and quality-of-model. However, some systematic discrepancies are also noted employing a Molprobity analysis, that are reproducible across multiple competition entries. A space of key refinement parameters is explored within ReMDFF to observe their impact within the final model. Choice of force field parameters and initial model seem to have the most significant impact on ReMDFF model-quality. To this end, very recently developed CHARMM36m force field parameters provide now more refined ReMDFF models than the ones originally submitted to the Cryo-EM challenge. Finally, a set of good-practices is prescribed for the community to benefit from the MDFF developments.
Conformational plasticity of the response regulator CpxR, a key player in Gammaproteobacteria virulence and drug-resistance.
Venue: Journal of structural biology

The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.
De novo main-chain modeling with MAINMAST in 2015/2016 EM Model Challenge
Venue: Journal of structural biology

Protein tertiary structure modeling is a critical step for the interpretation of three dimensional (3D) election microscopy density. Our group participated the 2015/2016 EM Model Challenge using the MAINMAST software for a de novo main chain modeling. The software generates local dense points using the mean shifting algorithm, and connects them into Cα models by calculating the minimum spanning tree and the longest path. Subsequently, full atom structure models are generated, which are subject to structural refinement. Here, we summarize the qualities of our submitted models and examine successful and unsuccessful models, including 3D models we did not submit to the Challenge. Our protocol using the MAINMAST software was sometimes able to build correct conformations with 3.4–5.1 Å RMSD. Unsuccessful models had failure of chain traces, however, their Cα positions and some local structures were quite correctly built. For evaluate the quality of the models, the MAINMAST software provides a confidence score for each Cα position from the consensus of top 100 scoring models.
Contributions of different modules of the plasminogen-binding Streptococcus pyogenes M-protein that mediate its functional dimerization.
Venue: Journal of structural biology

Group A Streptococcus pyogenes (GAS) is a causative agent of pharyngeal and dermal infections in humans. A major virulence determinant of GAS is its dimeric signature fibrillar M-protein (M-Prt), which is evolutionarily designed in modules, ranging from a hypervariable extracellular N-terminal region to a progressively more highly conserved C-terminus that is covalently anchored to the cell wall. Of the >250 GAS isolates classified, only the subset of skin-trophic Pattern D strains expresses a specific serotype of M-Prt, PAM, that directly binds to host human plasminogen (hPg) via its extracellular NH2-terminal variable A-domain region. This interaction allows these GAS strains to accumulate components of the host fibrinolytic system on their surfaces to serve extracellular functions. While structure-function studies have been accomplished on M-Prts from Pattern A-C GAS isolates with different direct ligand binding properties compared to PAM, much less is known regarding the structure-function relationships of PAM-type M-Prts, particularly their dimerization determinants. To examine these questions, PAMs from seven GAS strains with sequence variations in the NH2-terminal ligand binding domains, as well as truncated versions of PAM, were designed and studied. The results from bioinformatic and biophysical analyses show that the different domains of PAM are disparately engaged in dimerization. From these data, we propose an experimentally-based model for PAM secondary and quaternary structures that is highly dependent on the conserved helical C-terminal C-D-domains. In addition, while the N-terminal regions of PAMs are variable in sequence, the binding properties of hPg and its activated product, plasmin, to the A-domain, remain intact.
Map segmentation, automated model-building and their application to the Cryo-EM Model Challenge.
Venue: Journal of structural biology

A recently-developed method for identifying a compact, contiguous region representing the unique part of a density map was applied to 218 Cryo-EM maps with resolutions of 4.5 Å or better. The key elements of the segmentation procedure are (1) identification of all regions of density above a threshold and (2) choice of a unique set of these regions, taking symmetry into consideration, that maximize connectivity and compactness. This segmentation approach was then combined with tools for automated map sharpening and model-building to generate models for the 12 maps in the 2016 Cryo-EM Model Challenge in a fully automated manner. The resulting models have completeness from 24% to 82% and RMS distances from reference interpretations of 0.6 Å-2.1 Å.
High resolution single particle cryo-electron microscopy using beam-image shift.
Venue: Journal of structural biology

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.
Variability of the core geometry in parallel coiled-coil bundles.
Venue: Journal of structural biology

In protein modelling and design, an understanding of the relationship between sequence and structure is essential. Using parallel, homotetrameric coiled-coil structures as a model system, we demonstrated that machine learning techniques can be used to predict structural parameters directly from the sequence. Coiled coils are regular protein structures, which are of great interest as building blocks for assembling larger nanostructures. They are composed of two or more alpha-helices wrapped around each other to form a supercoiled bundle. The coiled-coil bundles are defined by four basic structural parameters: topology (parallel or antiparallel), radius, degree of supercoiling, and the rotation of helices around their axes. In parallel coiled coils the latter parameter, describing the hydrophobic core packing geometry, was assumed to show little variation. However, we found that subtle differences between structures of this type were not artifacts of structure determination and could be predicted directly from the sequence. Using this information in modelling narrows the structural parameter space that must be searched and thus significantly reduces the required computational time. Moreover, the sequence-structure rules can be used to explain the effects of point mutations and to shed light on the relationship between hydrophobic core architecture and coiled-coil topology.
A methodology using Gaussian-based density map approximation to assess sets of cryo-electron microscopy density maps.
Venue: Journal of structural biology

This article presents a methodology to assess a set of density maps, as used in the Blind Assessment Phase of the 2015/2016 Map Challenge (EMDataBank Validation Challenges). The synthetic and experimental cryo-electron microscopy (cryo-EM) density maps obtained by different single particle analysis protocols and by different participants, submitted in the Challenge Phase for assessment, were analyzed with this methodology and the obtained results are presented and discussed here. The goal of using such a methodology was to blindly identify the density maps with globally similar structural information, meaning the maps with the structural information mostly "reproduced" by different protocols. To this end, the density maps are "coarsened" using Gaussian-based approximations, with the same input approximation parameters for all maps of the target biological complex. The approximated maps are then represented in a common reduced-dimension (here, 3D) space of their correlation-coefficient-based distances, in which close maps mean similar maps. The distance analysis allows identifying maps with the most "reproduced" structural information by different protocols. The obtained results are also discussed taking into account the detailed information about the protocols that has been released after the end of the Blind Assessment Phase.
Studying intact bacterial peptidoglycan by proton-detected NMR spectroscopy at 100 kHz MAS frequency.
Venue: Journal of structural biology

The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeneity, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site-specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling.
Improved sample dispersion in cryo-EM using "perpetually-hydrated" graphene oxide flakes.
Venue: Journal of structural biology

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.
Map Challenge assessment: Fair comparison of single particle cryoEM reconstructions.
Venue: Journal of structural biology

Cryo-electron microscopy (cryoEM) is capable of achieving near-atomic resolution of biomolecular structures due to recent advances in hardware. Despite the long history of image processing software development for cryoEM, uncertainty about best practices and validation remains. The Map Challenge was therefore designed to test the current state of single particle reconstruction. As the first such challenge, the participants were given the freedom to analyze the cases in whichever way they wanted. Therefore, the maps submitted feature different sizes, sampling and orientations, making assessment non-trivial. To be fair, I developed a method to pose all maps in each case in the same configuration with minimal interpolation. I assessed the quality of these maps by visual inspection and Fourier shell correlation (FSC). Comparing the even-odd FSC with an FSC calculated against a reference structure analysis, I concluded that the quality of the maps related more to the user than to other factors, such as the software package used. Poor quality maps suffer either from lack of data or poor choices made by the user. Some maps appear significantly better than a reference or consensus of other maps, indicating overfitting. Best practices to avoid problems include an understanding of the effects of reference map modifications on particle image alignment, and generating appropriate masks. Ultimately, none of the issues revealed in the Map Challenge is insurmountable, as underscored by the excellent quality of reconstructions achieved by a significant number of participants.
Crystal structure of saposin D in an open conformation.
Venue: Journal of structural biology

Saposins are accessory proteins that aid in the degradation of sphingolipids by hydrolytic enzymes. Their structure usually comprises four α-helices arranged in various conformations including an open, V-shaped form that is generally associated with the ability to interact with membranes and/or enzymes to accentuate activity. Saposin D is required by the lysosomal hydrolase, acid ceramidase, which breaks down ceramide into sphingosine and free fatty acid, to display optimal activity. The structure of saposin D was previously determined in an inactive conformation, revealing a monomeric, closed and compact form. Here, we present the crystal structure of the open, V-shaped form of saposin D. The overall shape is similar to the open conformation found in other saposins with slight differences in the angles between the α-helices. The structure forms a dimer that serves to stabilize the hydrophobic surface exposed in the open form, which results in an internal, hydrophobic cavity that could be used to carry extracted membrane lipids.
A simple and robust procedure for preparing graphene-oxide cryo-EM grids.
Venue: Journal of structural biology

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and provide partial protection from the air-water interface. Additionally, the signal of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.
Evaluation system and web infrastructure for the second cryo-EM model challenge.
Venue: Journal of structural biology

An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail. It includes interactively sortable tables of evaluation scores for different subsets of models and different sublevels of structure organization, and a suite of visualization tools facilitating model analysis. The results are publicly accessible at http://model-compare.emdatabank.org.
Modulation of calcium oxalate dihydrate growth by phosphorylated osteopontin peptides.
Venue: Journal of structural biology

Osteopontin (OPN) is a significant component of kidney stone matrix and a key modulator of stone formation. Here, we investigated the effects of different phosphorylated states of a synthesized peptide of OPN (the ASARM peptide; acidic, serine- and aspartate-rich motif) on calcium oxalate dihydrate (COD) crystals, a major mineral phase of kidney stones. In vitro, phosphorylated OPN-ASARM peptides strongly inhibited COD crystal growth in solution as compared to the nonphosphorylated state, with increasing inhibitory potency correlating with the degree of peptide phosphorylation. Scanning electron microscopy revealed that the inhibition from the phosphopeptides resulted in distinctive, rosette-like crystal aggregates called spherulites. The OPN-ASARM peptides preferentially bound and specifically inhibited the {1 1 0} crystallographic faces of COD, as identified by combining atomic force microscopy and computational simulation approaches. These {1 1 0} surfaces of COD have high lattice calcium occupancy (exposure), providing preferential binding sites for the highly acidic peptides; binding and inhibition by OPN-ASARM peptides at the {1 1 0} faces led to crystal aggregation and intergrowth. The crystal spherulite formations obtained in vitro when using the most phosphorylated form of OPN-ASARM peptide at a high concentration, resembled crystal morphologies observed in vivo in a rat model of urolithiasis, in which crystal deposits in the kidney contain abundant OPN as revealed by immunogold labeling. A mechanistic model for spherulite formation is proposed based on the symmetry and crystallographic structure of COD, where the phosphate groups of OPN-ASARM bind to calcium atoms at [1 1 1] step risers on the COD {1 1 0} surface, inducing the periodic emergence of new COD crystals to form spherulites.
Reconstituting the formation of hierarchically porous silica patterns using diatom biomolecules.
Venue: Journal of structural biology

The genetically-controlled formation of complex-shaped inorganic materials by living organisms is an intriguing phenomenon. It illustrates our incomplete understanding of biological morphogenesis and demonstrates the feasibility of ecologically benign routes for materials technology. Amorphous SiO2 (silica) is taxonomically the most widespread biomineral, with diatoms, a large group of single-celled microalgae, being the most prolific producers. Silica is the main component of diatom cell walls, which exhibit species-specific patterns of pores that are hierarchically arranged and endow the material with advantageous properties. Despite recent advances in characterizing diatom biomolecules involved in biosilica morphogenesis, the mechanism of this process has remained controversial. Here we describe the in vitro synthesis of diatom-like, porous silica patterns using organic components that were isolated from biosilica of the diatom Cyclotella cryptica. The synthesis relies on the synergism of soluble biomolecules (long-chain polyamines and proteins) with an insoluble nanopatterned organic matrix. Biochemical dissection of the process revealed that the long-chain polyamines rather than the proteins are essential for efficient in vitro synthesis of the hierarchically porous silica patterns. Our results support the organic matrix hypothesis for morphogenesis of diatom biosilica and introduce organic matrices from diatoms as a new tool for the synthesis of meso- to microporous inorganic materials.
Optimal data-driven parameterization of coiled coils.
Venue: Journal of structural biology

α-Helical coiled coils (CCs) represent an important, highly regular protein folding motif. To date, many thousands of CC structures have been determined experimentally. Their geometry is usually modelled by theoretical equations introduced by F. Crick that involve a predefined set of parameters. Here we have addressed the problem of efficient CC parameterization from scratch by performing a statistical evaluation of all available CC structures. The procedure is based on the principal component analysis and yields a minimal set of independent parameters that provide for the reconstruction of the complete CC structure at a required precision. The approach is successfully validated on a set of canonical parallel CC dimers. Its applications include all cases where an efficient sampling of the CC geometry is important, such as for solving the phase problem in crystallography.
Single particle reconstruction and validation using Bsoft for the map challenge.
Venue: Journal of structural biology

The Bsoft package is aimed at processing electron micrographs for the determination of the three-dimensional structures of biological specimens. Recent advances in hardware allow us to solve structures to near atomic resolution using single particle analysis (SPA). The Map Challenge offered me an opportunity to test the ability of Bsoft to produce reconstructions from cryo-electron micrographs at the best resolution. I also wanted to understand what needed to be done to work towards full automation with validation. Here, I present two cases for the Map Challenge using Bsoft: ß-galactosidase and GroEL. I processed two independent subsets in each case with resolution-limited alignment. In both cases the reconstructions approached the expected resolution within a few iterations of alignment. I further validated the results by coherency-testing: i.e., that the reconstructions from real particles give better resolutions than reconstructions from the same number of aligned noise images. The key operations requiring attention for full automation are: particle picking, faster accurate alignment, proper mask generation, appropriate map sharpening, and understanding the amount of data needed to reach a desired resolution.