nils-herrmann/clean_scirepeval huggingface dataset:
Structural flexibility in the Helicobacter pylori peptidyl-prolyl cis,trans-isomerase HP0175 is achieved through an extension of the chaperone helices.
Venue: Journal of structural biology

Helicobacter pylori infects the gastric epithelium of half the global population, where infections can persist into adenocarcinomas and peptic ulcers. H. pylori secretes several proteins that lend to its pathogenesis and survival including VacA, CagA, γ-glutamyltransferase and HP0175. HP0175, also known as HpCBF2, classified as a peptidyl-prolyl cis,trans-isomerase, has been shown to induce apoptosis through a cascade of mechanisms initiated though its interaction with toll like receptor 4 (TLR4). Here, we report the structure of apo-HP0175 at 2.09 Å with a single monomer in the asymmetric unit. Chromatographic, light scattering and mass spectrometric analysis of HP0175 in solution indicate that the protein is mainly monomeric under low salt conditions, while increasing ionic interactions facilitates protein dimerization. A comparison of the apo-HP0175 structure to that of the indole-2-carboxylic acid-bound form shows movement of the N- and C-terminal helices upon interaction of the catalytic residues in the binding pocket. Helix extension of the N/C chaperone domains between apo and I2CA-bound HP0175 supports previous findings in parvulin PPIases for their role in protein stabilization (and accommodation of variable protein lengths) of those undergoing catalysis.
Elemental composition and ultrafine structure of the skeleton in shell-bearing protists-A case study of phaeodarians and radiolarians.
Venue: Journal of structural biology

Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radiolarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method presented in this study could be a useful approach to examine the chemical and structural characteristics of unculturable and/or rare microorganisms.
Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain.
Venue: Journal of structural biology

In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.
A new algorithm for high-resolution reconstruction of single particles by electron microscopy.
Venue: Journal of structural biology

The Map Challenge organized by the Electron Microscopy Data Bank has prompted the development of an Xmipp high resolution reconstruction protocol (which we will refer to as highres) that is integrated in the software platform Scipion. In this work we describe the details of the image angular alignment and map reconstruction steps in our new method. This algorithm is similar to the standard projection matching approach with some important modifications, especially in the area of detecting significant features in the reconstructed volume. We show that the new method is able to produce higher resolution maps than the current de facto standard as measured by the Fourier Shell Correlation, the Monogenic Local Resolution and EMRinger.
Assessment of structural features in Cryo-EM density maps using SSE and side chain Z-scores.
Venue: Journal of structural biology

We introduce a new method for assessing resolvability of structural features in density maps from Cryo-Electron Microscopy (Cryo-EM) using fitted or derived models. It calculates Z-scores for secondary structure elements (SSEs) and side chains. Z-scores capture how much larger the cross-correlation score (CCS) is for atoms in such features at their placed locations compared to the CCS at displaced positions. Z-scores are larger when the structural features are well-resolved, as confirmed by visual analysis. This method was applied to all 66 maps submitted to the 2015/2016 EMDB map challenge. For each map, the fitted model provided by the map committee was used in this assessment. The average Z-scores for each map and fitted model correlate moderately well with reported map resolutions (r2 = 0.45 for SSE Z-scores and r2 = 0.56 for side chain Z-scores). Rankings of the submitted maps based on average Z-scores seem to more closely agree with visual analysis. Z-scores can also be used to pinpoint which parts of a model are well-resolved in a map, and which parts of the model may need further fitting or refinement to make the model better match the density.
APPLE Picker: Automatic Particle Picking, a Low-Effort Cryo-EM Framework
Venue: Journal of structural biology

Particle picking is a crucial first step in the computational pipeline of single-particle cryo-electron microscopy (cryo-EM). Selecting particles from the micrographs is difficult especially for small particles with low contrast. As high-resolution reconstruction typically requires hundreds of thousands of particles, manually picking that many particles is often too time-consuming. While template-based particle picking is currently a popular approach, it may suffer from introducing manual bias into the selection process. In addition, this approach is still somewhat time-consuming. This paper presents the APPLE (Automatic Particle Picking with Low user Effort) picker, a simple and novel approach for fast, accurate, and template-free particle picking. This approach is evaluated on publicly available datasets containing micrographs of β-galactosidase, T20S proteasome, 70S ribosome and keyhole limpet hemocyanin projections.
Structural behavior of keratin-associated protein 8.1 in human hair as revealed by a monoclonal antibody.
Venue: Journal of structural biology

Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.
Structural and functional analyses of calcium ion response factors in the mantle of Pinctada fucata.
Venue: Journal of structural biology

The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.
The analysis of subtle internal communications through mutation studies in periplasmic metal uptake protein CLas-ZnuA2.
Venue: Journal of structural biology

The subtle internal communications through an intricate network of interactions play a key role in metal-binding and release in periplasmic metal uptake proteins of cluster A-I family, a component of ABC transport system. These proteins have evolved different mechanisms of metal-binding and release through sequence and thereby structure-function divergence. The CLas-ZnuA2 from Candidatus Liberibacter asiaticus (CLA), in previous studies, showed a lower metal-binding affinity. The subtle communications within and between domains from crystal structure analysis revealed that protein seems to prefer a metal-free state. The unique features of CLas-ZnuA2 included a highly restrained loop L3 and presence of a proline in linker helix. In present work, S38A and Y68F mutants were studied as they play an important role during metal-binding in CLas-ZnuA2. The mutations in linker helix could not be studied as the expressed protein was not soluble and in most cases degraded with time. The crystal structure analysis of (S38A and Y68F) mutants in metal-free and metal-bound forms showed variations in interactions, an increase in number of alternate conformations and distortions in secondary structure elements, despite a similar overall structure, suggesting alterations in internal communications. The results suggested that any change in critical residues could alter the subtle internal communications and result in disturbing the fine-tuned structure required for optimal functioning.
Conformational flexibility of pore loop-1 gives insights into substrate translocation by the AAA+ protease FtsH.
Venue: Journal of structural biology

Two crystal structures of a transmembrane helix-lacking FtsH construct from Aquifex aeolicus have been determined at 2.9 Å and 3.3 Å resolution in space groups R32 and P312, respectively. Both structures are virtually identical despite different crystal packing contacts. In both structures, the FtsH hexamer is created from two different subunits of the asymmetric unit by the threefold symmetry of the crystals. Similar to other published structures, all subunits are loaded with ADP and the two subunit in the asymmetric unit resemble the already known open and closed conformations. Within the ATPase cycle while the whole subunit switches from the opened to the closed state, pore loop-1 interacts with the substrate and translocates it into the proteolytic chamber. Unique to our models is a presumably inactive conformation of the pore loop which allows the closed conformation to switch back to the opened state without pushing the substrate out again. Our structures give further insights on how this new pore loop conformation is induced and how it is linked to the intersubunit signalling network.
Building atomic models based on near atomic resolution cryoEM maps with existing tools.
Venue: Journal of structural biology

The EMDataBank Validation Challenge was a challenging task for students newly introduced to the cryoEM and molecular modeling fields. However, the competition provided an effective space for student modelers to discover and explore the potentials of atomic modeling and refinement by practicing on published atomic structures. Here, by employing manual molecular modeling programs such as Coot, Phenix, and Chimera, we have regularized and improved three targets. The T20S proteasome and TRPV1 ion channel allowed us to broaden our understanding of these modeling techniques while the 70S ribosome served as a challenge to test the limits of our abilities. We were successful in our efforts to improve each of the models and provide here our cohesive methodology for de novo modeling with and without homology models, which may serve as a starting point for other undergraduates and researchers just entering the realm of cryoEM. Additionally, we provide some constructive criticism to facilitate the introduction of said undergraduates and researchers into cryoEM in the future.
The first single particle analysis Map Challenge: A summary of the assessments.
Venue: Journal of structural biology

The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.
The 3-D structure of VNG0258H/RosR - A haloarchaeal DNA-binding protein in its ionic shell.
Venue: Journal of structural biology

Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus make significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences, with implications to protein-DNA interactions in hyper-saline environments.
3D micro structural analysis of human cortical bone in paired femoral diaphysis, femoral neck and radial diaphysis.
Venue: Journal of structural biology

Human bone is known to adapt to its mechanical environment in a living body. Both its architecture and microstructure may differ between weight-bearing and non-weight-bearing bones. The aim of the current study was to analyze in three dimensions, the morphology of the multi-scale porosities on human cortical bone at different locations. Eight paired femoral diaphyses, femoral necks, and radial diaphyses were imaged using Synchrotron Radiation µCT with a 0.7 µm isotropic voxel size. The spatial resolution facilitates the investigation of the multiscale porosities of cortical bone, from the osteonal canals system down to the osteocyte lacunar system. Our results showed significant differences in the microstructural properties, regarding both osteonal canals and osteocytes lacunae, between the different anatomical locations. The radius presents significantly lower osteonal canal volume fraction and smaller osteonal canals than the femoral diaphysis or neck. Osteocytes lacunae observed in the radius are significantly different in shape than in the femur, and lacunar density is higher in the femoral neck. These results show that the radius, a non-weight-bearing bone, is significantly different in terms of its microstructure from a weight-bearing bone such as the femur. This implies that the cortical bone properties evaluated on the femoral diaphysis, the main location studied within the literature, cannot be generalized to other anatomical locations.
Assessment of detailed conformations suggests strategies for improving cryoEM models: Helix at lower resolution, ensembles, pre-refinement fixups, and validation at multi-residue length scale.
Venue: Journal of structural biology

We find that the overall quite good methods used in the CryoEM Model Challenge could still benefit greatly from several strategies for improving local conformations. Our assessments primarily use validation criteria from the MolProbity web service. Those criteria include MolProbity's all-atom contact analysis, updated versions of standard conformational validations for protein and RNA, plus two recent additions: first, flags for cis-nonPro and twisted peptides, and second, the CaBLAM system for diagnosing secondary structure, validating Cα backbone, and validating adjacent peptide CO orientations in the context of the Cα trace. In general, automated ab initio building of starting models is quite good at backbone connectivity but often fails at local conformation or sequence register, especially at poorer than 3.5 Å resolution. However, we show that even if criteria (such as Ramachandran or rotamer) are explicitly restrained to improve refinement behavior and overall validation scores, automated optimization of a deposited structure seldom corrects specific misfittings that start in the wrong local minimum, but just hides them. Therefore, local problems should be identified, and as many as possible corrected, before starting refinement. Secondary structures are confusing at 3-4 Å but can be better recognized at 6-8 Å. In future model challenges, specific steps being tested (such as segmentation) and the required documentation (such as PDB code of starting model) should each be explicitly defined, so competing methods on a given task can be meaningfully compared. Individual local examples are presented here, to understand what local mistakes and corrections look like in 3D, how they probably arise, and what possible improvements to methodology might help avoid them. At these resolutions, both structural biologists and end-users need meaningful estimates of local uncertainty, perhaps through explicit ensembles. Fitting problems can best be diagnosed by validation that spans multiple residues; CaBLAM is such a multi-residue tool, and its effectiveness is demonstrated.
Rapid near-atomic resolution single-particle 3D reconstruction with SIMPLE.
Venue: Journal of structural biology

Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.