Open-access and free articles in Acta Crystallographica Section D: Structural Biology
Acta Crystallographica Section D: Structural Biology welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function articles that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such articles should present new insights into biology, chemistry or structure. Articles on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Articles on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability.
Copyright (c) 2026 International Union of Crystallography
Open-access and free articles in Acta Crystallographica Section D Structural Biology
Acta Crystallographica Section D Structural Biology
Crystal structure of Schistosoma mansoni cathepsin D1 in complex with a nanobody reveals the conformation of the propeptide-bound state
Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme–nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.
SCHISTOSOMA MANSONI; CATHEPSIN D; NANOBODIES; X-RAY CRYSTALLOGRAPHY
Acta Crystallographica Section D: Structural Biology
Explicit correction of severely non-uniform distributions of cryo-EM views
The quality of three-dimensional macromolecular image reconstruction by cryo electron microscopy (cryo-EM) strongly depends on the number and the quality of the respective two-dimensional projections and on their angular distribution in space. Distributions with one or a few strongly preferred particle orientations may result in maps that are deformed in certain directions. A simple removal of overrepresented views may improve the quality of the reconstructed maps when the level of noise in the two-dimensional (2D) projections is low and the data-set size can afford this removal, but is counterproductive otherwise. Complementarily, giving an increased weight to underrepresented views, or taking multiple copies of them during the reconstruction, may improve the results, naturally, depending on how non-uniform the view distribution is. This work describes the results of three-dimensional (3D) reconstructions using an explicit correction of the number of overrepresented and underrepresented projections for non-uniformly distributed sets. Such correction can be considered as a potential preprocessing, fast and simple, during 3D reconstruction in the image-processing and cryo-EM structure-determination workflow.
CRYO-EM; 3D RECONSTRUCTION; VIEW FREQUENCY; NON-UNIFORM VIEW DISTRIBUTION; PREFERRED ORIENTATIONS; DISTRIBUTION CORRECTION; RECONSTRUCTION IMPROVEMENT; EXPLICIT DISTRIBUTION HOMOGENIZATION; IMAGE IMPROVEMENT
Associating protein residues in the literature with structural data
Protein structures are crucial in understanding the function, mechanism and disease-causing variants of proteins within any living cell. A number of experimental techniques are employed by researchers to determine such structures. Through structure inspection in molecular viewers, combined with supporting biochemical and biophysical experiments, scientists are able to identify the function of a protein, its reaction mechanism and effects caused by sequence variation. These detailed findings, supported by experimental results, are documented by being described in the scientific literature and by making the accompanying data open source. However, it has become increasingly difficult for a reader, in particular a non-expert, to access the correct additional information and assess the validity of the conclusions drawn based on experimental results. A reader is often required to resort to a number of different software packages to access the different data types. Here, we present a first-of-its-kind implementation of an artificial intelligence- and text-mining-supported software tool that allows the association of mentions in the text of one or more specific protein residues with their corresponding counterparts in the respective protein structure or structures. Our application allows a researcher to explore a residue of interest in the context of a publication and its respective protein structure, supported by its experimental evidence, in a single view. We describe model implementation, annotation extraction, downstream processing, dissemination and visualization at the IUCr and PDBe. The application presented is primarily aimed at readers of IUCr publications and users visiting the PDBe entry pages. However, we believe that in the future our application will be a valuable tool for reviewers of new submissions to IUCr journals and may even be useful as a curation tool involving the authors of a publication as annotation validators.
TEXT MINING; LARGE LANGUAGE MODELS; TEXT ANNOTATIONS; KNOWLEDGE LINKING; PROTEIN STRUCTURE
Strategies for mitigating radiation damage and improving data completeness in 3D electron diffraction of protein crystals
While 3D electron diffraction (3D-ED or microcrystal electron diffraction; MicroED) has emerged as a promising method for protein structure determination, its applicability is hindered by a high susceptibility to radiation damage, leading to a decreasing signal-to-noise ratio in consecutive diffraction patterns that limits the quality (resolution and redundancy) of the data. In addition, data completeness may be restricted due to the geometrical limitations of current sample holders and stages. Although specialized equipment can overcome these challenges, many laboratories do not have access to such instrumentation. In this work, we introduce an approach that addresses these issues using a commonly available 200 keV cryo-electron microscope. The multi-position acquisition technique that we present here combines (i) multiple data acquisitions from a single crystal over several tilt ranges and (ii) merging data from a small number of crystals each tilted about a different axis. The robustness of this approach is demonstrated by the de novo elucidation of a protein–peptide complex structure from only two orthorhombic microcrystals.
ELECTRON DIFFRACTION; RADIATION DAMAGE; DATA COMPLETENESS; 3D-ED; MICROED
Towards patient-relevant structures: reviewing body-temperature biological macromolecules and their ligands for pharmaceutical applications
The aim of our structures for eventual clinical application is to be relevant. Regulation of pharmaceutical lead compounds, however, does not yet involve the need for patient-relevant macromolecular structures determined at 37°C, as it is not yet known whether crystal growth and diffraction at 37°C versus standard cryo-condition practices will reveal significant binding variations applicable for drug development or, in the case of extremophiles, provide insight into their function. However, for select examples in the literature interesting changes occurred, and support the initiative that data collection at high temperatures should be considered. This topical review considers a Protein Data Bank (PDB) and Cambridge Structural Database (CSD) data survey of crystal structures that have been determined at elevated temperatures, i.e. neither under cryogenic conditions nor at typical room-temperature conditions of 20–25°C, and reveals a few hurdles as well as many successes in reaching such patient-relevant structures. It highlights key methodology that appears in the literature which could benefit those considering related research. Since it is possible for crystallographic structure-determination methods to be adapted to 37°C, amid some challenges, we encourage the initiative that many more could be determined at 37°C. Included in the studies deposited in the PDB are some that have been performed at temperatures in excess of >37°C, and surprisingly several at even higher temperatures (i.e. 50–90°C). The overall aim of determining the 3D structure of a biological macromolecule at its natural body temperature has in principle to include crystallization and diffraction data collection. In the survey we find very few crystallizations performed at 37°C followed by data collection at the same temperature, and few have conducted a systematic study of comparing the changes occurring at 100 K versus 37°C. It is of course assumed that some key drug binding in proteins may occur over a narrow temperature range appropriate for mesophilic organisms, whereas for thermophilic organisms the protein may well exist over a wide temperature range reflecting that in which the organism is able to thrive. For the higher temperature structure solutions, those in the range which is more appropriate for thermophiles or hyperthermophiles, no crystallizations at these extreme temperatures have yet been conducted. The ability to conduct crystallization at 37°C and obtain acceptable high-resolution data at the same temperature is surely encouraging to the crystallographic community to build on these achievements for this and the full temperature range. We describe aspects of crystallization, mounting and transfer of crystals, data collection, reporting of metadata within databases etc. that have been notable during the survey of the data and highlight them here for the benefit of the community which may be considering 37°C data analysis from pre-crystal growth to re-refinement of data. For comparable data and to avoid any experimental bias, we also encourage the community to complete the analysis sequentially, as few have considered this holistic analysis of solid-state variations which may occur over the low-to-high temperature range.
BODY TEMPERATURE; PHYSIOLOGICAL CONDITIONS; 37DEGREESC; DIFFRACTION STRUCTURAL BIOLOGY; PDB-CSD SURVEY; THERMOPHILES; HYPERTHERMOPHILES
Leopard-EM: an extensible 2D template-matching package to accelerate in situ structural biology
The ability to generate high-resolution views of cells with cryogenic electron microscopy (cryo-EM) can reveal the molecular mechanisms of biological processes in their native cellular context. The revolutionary impact of this strategy is limited by the difficulty of accurately annotating structures within these images. 2D template matching (2DTM), in which high-resolution structural models are used as computational probes to locate and orient molecular complexes with high precision, has shown initial promise in annotating single molecules in cellular cryo-EM images. While the scientific community works to identify best practices for applying 2DTM to specific biological questions and to maximize sensitivity and throughput, a modular and extensible software architecture would support the rapid development of novel methodological approaches, thus accelerating innovation within the field. To achieve this, we developed Leopard-EM (Location and orientation of particles found using two-dimensional tEmplate Matching), a modular Python-based 2DTM implementation built to be easily customizable. We implemented an automated pixel-size refinement procedure and find that 2DTM is sensitive to pixel-size errors of ∼0.2%. To demonstrate the flexibility of the Leopard-EM architecture, we developed a constrained search protocol that improved small ribosomal subunit detection by approximately eightfold by using initial locations and orientations determined for the large ribosomal subunit. Using this strategy, we captured a distribution of ribosome rotation states within the living cell at single-molecule resolution. We envision that Leopard-EM can be used as a platform for the development of in situ cryo-EM data-processing workflows, facilitating the rapid development of this field. Leopard-EM is available at https://github.com/Lucaslab-Berkeley/Leopard-EM.
IN SITU STRUCTURAL BIOLOGY; CRYO-EM; CRYO-ET; TEMPLATE MATCHING; STRUCTURAL CELL BIOLOGY
Completion of partial structures using Patterson maps with the CrysFormer machine-learning model
Protein structure determination has long been one of the primary challenges of structural biology, to which deep machine learning (ML)-based approaches have increasingly been applied. However, these ML models generally do not directly incorporate the experimental measurements, such as X-ray crystallographic diffraction data. To this end, we explore an approach that more tightly couples these traditional crystallographic and recent ML-based methods by training a hybrid 3D vision transformer and convolutional network on inputs from both domains. We make use of two distinct input constructs: Patterson maps, which are directly obtainable from crystallographic data, and `partial structure' template maps derived from predicted structures deposited in the AlphaFold Protein Structure Database with subsequently omitted residues. With these, we predict electron-density maps that are then post-processed into atomic models through standard crystallographic refinement processes. Introducing an initial data set of small protein fragments taken from Protein Data Bank entries and placing them in hypothetical crystal settings, we demonstrate that our method is effective at both improving the phases of the crystallographic structure factors and completing the regions missing from partial structure templates, as well as improving the agreement of the electron-density maps with the ground-truth atomic structures.
PHASING; PATTERSON MAPS; MACHINE LEARNING; STRUCTURE DETERMINATION; X-RAY CRYSTALLOGRAPHY
Xtricorder: a likelihood-enhanced self-rotation function and application to a machine learning-enhanced Matthews prediction of asymmetric unit copy number
Analysis of crystallographic diffraction data after collection and integration but before phasing gives the crystallographer a `first-look' assessment of data quality and flags potential challenges in subsequent structure determination. We here report the development of Xtricorder, a `first-look' application specifically targeted at likelihood-based phasing. Xtricorder incorporates the full array of analyses previously available in the Phaser codebase, with some enhancements and updates, in a more streamlined and accessible implementation. In addition, Xtricorder offers a likelihood-enhanced self-rotation function. A novel graphical representation of the self-rotation function, the `composite-section diagram', presents the results for user inspection and has the added advantage that, in an adapted form, it is appropriate for training a convolutional neural network to enhance the standard Matthews analysis and double the accuracy of asymmetric unit copy-number prediction. We investigate the usefulness of the likelihood-enhanced self-rotation function in `first-look' analyses, exploring the circumstances under which the self-rotation function results are useful, and discuss the application to AI-generated structure prediction.
MOLECULAR REPLACEMENT; MATTHEWS COEFFICIENT; MACHINE LEARNING; PHASER; PHASERTNG; SELF-ROTATION FUNCTION
Polo-like kinase 1–inhibitor co-complex structures via the surface-entropy reduction approach and a DARPin-assisted approach
Polo-like kinase 1 (PLK1) is a major regulator of cell division and has been pursued as a drug target for cancer therapy for a long time. Crystallization of the kinase domain has proven to be exceptionally challenging. Previously, we published a crystallization approach using a PLK1-specific designed ankyrin-repeat protein (DARPin) as a crystallization facilitator. Here, we report an alternative route: crystallization was successful after the introduction of a double mutation which reduced surface entropy and enabled the formation of a new crystal contact. This new PLK1 crystallization system was used to determine the first co-complex crystal structure of the Bayer thiazolidinone lead series, as well as crystal structures with representatives of two competitor inhibitor series. The molecular binding modes of these three inhibitors are analysed and discussed, and the surface-entropy reduction approach is compared with the surface modifications employed by us and others to enable the crystallization of PLK1.
SURFACE-ENTROPY REDUCTION; SER; KINASE; CRYSTALLIZATION; PLK1 SMALL-MOLECULE INHIBITOR
Exploration of questionable backbone conformations in crystallographic structure models using a structural alphabet
More than 80% of protein structure models in the Protein Data Bank have been solved using X-ray crystallography. Despite continuous improvements in this experimental technique, crystallographic structure models may still present artifacts related to the crystallization process as well as errors introduced during model building and refinement, even in high-resolution cases. Such limitations can alter atomic or residue positions, leading to local misconformations, local or domain rearrangements, and occasionally global distortions. In this study, we developed a protocol to locate residues with questionable conformations, where conformations may be uncertain, atypical or influenced by crystallographic modeling and refinement. To do so, we started from a set of 826 nonredundant X-ray protein structure models. Each X-ray model underwent an energy-minimization step that relaxes atomic geometry by reducing potential energy. Residues that exhibited different local conformations between the X-ray and minimized models were therefore considered as having questionable conformations. To identify them, we compared the X-ray and minimized models of each protein using the HMM-SA structural alphabet. Our results revealed that over 18% of the residues in the protein set have questionable conformations in their backbone. These conformations can occur either as isolated events within the protein sequence or can form patterns. Moreover, we observed that the frequency of questionable conformations per X-ray model was independent of factors such as the date of deposition, resolution or crystal system. Analysis of the properties of residues associated with questionable conformations revealed that they do not specifically occur in flexible or accessible regions. However, there is a correlation between questionable conformations and secondary structures, with a particular overrepresentation of residues with questionable conformations in α-helices. We then further investigated questionable conformations in the structure model of ligand-free HIV-2 protease (PR2). By combining our questionable conformation-detection protocol with molecular-dynamics simulation, we demonstrated that approximately half of the questionable conformations in PDB entry 1hsi correspond to local conformations that are sparsely sampled by PR2 during the molecular-dynamics simulation or are structural outliers detected by the wwPDB report. In addition, our results suggested that these questionable conformations may affect the position of the flaps, two β-sheets forming the top of the binding site. In PDB entry 1hsi, their relative arrangement appears atypical compared with MD simulations, raising questions about the biological relevance of this conformation. To conclude, we have developed a protocol to quantify and localize questionable backbone conformations in X-ray structure models, which can affect the interpretation of structural data.
QUESTIONABLE CONFORMATIONS; CRYSTALLOGRAPHIC STRUCTURES; STRUCTURAL ALPHABET; STRUCTURAL DEFORMATION
Limiting the effects of radiation damage in MicroED through dose selection during data processing
Microcrystal electron diffraction (MicroED), also known as three-dimensional electron diffraction (3D ED), allows the collection of diffraction data from submicrometre-sized crystals under low electron-dose conditions. Despite having several advantages over conventional X-ray crystallographic techniques, susceptibility to radiation damage is a great challenge that remains to be solved in MicroED. Similar to X-ray crystallography, radiation damage to the macromolecular crystal structures in MicroED manifests in two forms: global damage that affects the overall order of the crystal lattice and site-specific damage that affects highly sensitive residues and moieties in macromolecules. Traditionally, the unit e− Å−2 has been used for electron-dose estimations, which does not consider the interaction between the incident electron beam and the sample. In this study, we clarify the terminology for describing `dose' in electron crystallography, including the procedure for converting values from e− Å−2 to grays (Gy). Furthermore, we investigated data-processing strategies that could be used to limit the effects of radiation damage to the crystal. During MicroED data collection, radiation damage increases with the number of acquired ED frames because the accumulated electron dose increases. Data collected from several crystals and processed in this way can be merged to increase the completeness and subsequently be used for structure refinement. According to our results, this approach improves the resolution of the data, the data statistics, the structure determination and the quality of the final structure.
MICROCRYSTAL ELECTRON DIFFRACTION; MICROED; DATA PROCESSING; MACROMOLECULAR STRUCTURE; RADIATION DAMAGE
A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the Limosilactobacillus reuteri accessory secretion system
The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC53608 from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC100–23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC53608 became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.
ACCESSORY SECRETION SYSTEMS; GLYCOSYLTRANSFERASES; CRYSTAL STRUCTURE; GTFC
Perspective on structure predictions of disorder
ALPHAFOLD2; DISORDER PREDICTION; LOW PLDDT
Crystallographic fragment screening against SARS-CoV-2 nonstructural protein 1 using the F2X-Entry Screen and a newly developed fragment library
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. This underpins the need for novel therapeutics against this virus. Nonstructural protein 1 (Nsp1) of SARS-CoV-2 is a multifunctional protein with an essential role in viral replication. As such, it presents itself as an attractive target for drug discovery. Here, we describe two crystallographic fragment-screening campaigns against Nsp1, one using the established F2X-Entry Screen and one using a new, chemically and structurally diverse fragment library, which we call the KIT library. Together, 21 hits could be identified from 192 screened fragments, which constitutes the highest hit rate reported for Nsp1 to date. Many hits bind to a key functional region and interact with residues involved in cellular mRNA cleavage, ribosome binding and viral RNA recognition. Furthermore, most of the identified fragments share a common binding mode, providing promising starting points for further optimization into drug-like compounds that can disrupt the role of Nsp1 in viral replication.
X-RAY CRYSTALLOGRAPHY; CRYSTALLOGRAPHIC FRAGMENT SCREENING; SARS-COV-2; NONSTRUCTURAL PROTEIN 1; F2X-ENTRY LIBRARY
Similarity between two periodic functions is commonly assessed by comparing their Fourier coefficients within resolution shells. In particular, this approach is widely used in both crystallography and cryo-electron microscopy (cryoEM). The definition of these shells, that is the choice of resolution scale for their boundaries, can be guided by the specific goals of the analysis, by the expected features of the studied functions or simply by convention. In cryoEM, shell boundaries are traditionally defined uniformly in inverse resolution. This convention results in a vast imbalance in the number of Fourier coefficients per shell, which may bias statistical comparisons and can make function plots misleading. Constructing resolution shells with approximately equal numbers of Fourier coefficients can be achieved automatically by defining shell boundaries uniformly on the inverse cubic resolution scale. This transformation effectively zooms into the high-resolution region, which is typically the primary focus of analysis. For Fourier shell correlation (FSC) calculations between half-maps, the characteristic sigmoidal curves were observed to transform into profiles that permit piecewise linear interpolation, which may make FSC analysis more robust.
FSC CURVES; RESOLUTION SHELLS; INVERSE CUBIC RESOLUTION; UNIFORMLY FILLED SHELLS; HALF-MAPS; PIECEWISE LINEAR APPROXIMATION
Critical evaluation of three cryo-EM structures of particulate methane monooxygenase by quantum refinement
Particulate methane monooxygenase (pMMO) is an enzyme that converts methane into methanol at ambient temperature and pressure. Over the past three decades, the metal content and location of the active site have been highly controversial. Recent single-particle cryogenic electron-microscopy (cryo-EM) structures have furthered this debate. In this study, three cryo-EM structures (PDB entries 7s4h, 7s4j and 7ev9) are analysed by quantum refinement (QR). This approach augments traditional structural refinement with quantum-mechanical (QM) calculations for a small but interesting part of the protein (in this case, the copper sites). Our results indicate that the bis-His (CuA) site is correctly modelled as a mononuclear copper site in all three structures. The His-brace (CuB) site is also best modelled as mononuclear in all structures, although it was suggested to be a binuclear site in PDB entry 7ev9. The CuC site, which is observed only in PDB entry 7s4j, is correctly modelled and is probably reduced in the structure. The CuD putative active site, observed only in PDB entry 7s4h, is also mononuclear, but a water molecule might at least intermittently coordinate to the copper ion. On the other hand, our study does not find any support for the five additional copper ions suggested to be present in PDB entry 7ev9, including the suggested trinuclear active site and two sites in the so-called copper sponge. Instead, more chemically reasonable structures and better fit to both the cryo-EM and QM data are obtained if these copper ions are replaced with water molecules. This study illustrates the potential of QR as a standard component of cryo-EM studies for metal sites, for which reliable empirical restraints are missing.
PARTICULATE METHANE MONOOXYGENASE; CRYOGENIC ELECTRON-MICROSCOPY; QUANTUM REFINEMENT; COPPER
Cryo-EM single-particle analysis expanding towards increasingly native samples
The explosion of cryo-electron microscopy (cryo-EM) over the last decade has brought with it a range of new approaches for gaining high-resolution structural information on previously inaccessible biological systems. Cryo-EM single-particle analysis (SPA) approaches typically entail overexpression and purification of the target protein. Larger and more complex molecular assemblies often require extensive optimization of the expression, purification and reconstitution procedures. Additionally, prior knowledge of the composition of the structure of interest is required. Approaches employing cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET) have proven incredibly useful for exploring protein structures within cells while maintaining near-native conditions. Such strategies avoid purification of the target protein or protein complex, yet are often still limited in throughput and achievable resolution. Here, we highlight recent studies demonstrating that the range of samples suitable for SPA is expanding towards increasingly more native samples. We specifically focus on studies investigating complex macromolecular assemblies where tailored sample-preparation strategies made them amenable for SPA, while still keeping them in close-to-native conditions. These examples show that SPA has become a discovery tool for de novo protein identification and complex stoichiometry in more complex and thicker samples.
SINGLE-PARTICLE ANALYSIS; CRYO-EM; CRYO-ET
Reconstructing biological molecules with help from video gamers
Foldit is a citizen science video game in which players tackle a variety of complex biochemistry puzzles. Here, we describe a new series of puzzles in which Foldit players improve the accuracy of models in the public repository of experimental protein structure models, the Protein Data Bank (PDB). Analyzing the results of these puzzles showed that the Foldit players were able to considerably improve the deposited structures. We describe a mechanism by which the efforts of the Foldit players can be fed back into the structural biology scientific record by using Foldit results as improved input for the PDB-REDO databank. These efforts highlight the continued need for the engagement of the lay population in science.
FOLDIT; PDB-REDO; CITIZEN SCIENCE; MODEL CURATION
How many (distinguishable) classes can we identify in single-particle analysis?
Heterogeneity in cryoEM is essential for capturing the structural variability of macromolecules, reflecting their functional states and biological significance. However, estimating heterogeneity remains challenging due to particle mis­classification and algorithmic biases, which can lead to reconstructions that blend distinct conformations or fail to resolve subtle differences. Furthermore, the low signal-to-noise ratio inherent in cryo-EM data makes it nearly impossible to detect minute structural changes, as noise often obscures subtle variations in macromolecular projections. In this paper, we investigate the use of p-values associated with the null hypothesis that the observed classification differs from a random partition of the input data set, thereby providing a statistical framework for determining the number of distinguishable classes present in a given data set.
CRYO-ELECTRON MICROSCOPY; 3D CLASSIFICATION; STRUCTURAL HETEROGENEITY; STATISTICAL SIGNIFICANCE; REPRODUCIBILITY ANALYSIS
Categorizing prediction modes within low-pLDDT regions of AlphaFold2 structures: near-predictive, pseudostructure and barbed wire
AlphaFold2 protein structure predictions are widely available for structural biology uses. These predictions, especially for eukaryotic proteins, frequently contain extensive regions predicted below the pLDDT = 70 level, the rule-of-thumb cutoff for high confidence. This work identifies major modes of behavior within low-pLDDT regions through a survey of human proteome predictions provided by the AlphaFold Protein Structure Database. The near-predictive mode resembles folded protein and can be a nearly accurate prediction. Barbed wire is extremely unprotein-like, being recognized by wide looping coils, an absence of packing contacts and numerous signature validation outliers, and it represents a region where the conformation has no predictive value. Pseudo­structure presents an intermediate behavior with a misleading appearance of isolated and badly formed secondary-structure-like elements. These prediction modes are compared with annotations of disorder from MobiDB, showing general correlation between barbed wire/pseudostructure and many measures of disorder, an association between pseudostructure and signal peptides, and an association between near-predictive and regions of conditional folding. To enable users to identify these regions within a prediction, a new Phenix tool is developed encompassing the results of this work, including prediction annotation, visual markup and residue selection based on these prediction modes. This tool will help users develop expertise in interpreting difficult AlphaFold predictions and identify the near-predictive regions that can aid in molecular replacement when a prediction does not contain enough high-pLDDT regions.
ALPHAFOLD2; STRUCTURE PREDICTION; CONDITIONAL FOLDING; STRUCTURE VALIDATION; INTRINSICALLY DISORDERED REGIONS; LOW PLDDT; BARBED WIRE; NEAR-PREDICTIVE; SIGNAL PEPTIDES
Validation of helical symmetry parameters in the EMDB
Helical symmetry is a structural feature of many biological assemblies, including cytoskeletons, viruses and pathological amyloid fibrils. The helical parameters twist and rise are unique metadata for helical structures. With the increasing number of helical structures being resolved through cryo-EM and deposited in the EMDB, there is a growing possibility of errors in the metadata associated with these entries. During our cryo-EM analysis of protein amyloids and the development of helical analysis tools, we realized that many deposited helical parameters appear to be inconsistent with the associated density maps. Here, we have developed a comprehensive validation process that examines the consistency of these parameters by combining high-throughput computational evaluation with manual verification. Multiple errors were identified and corrected for ∼14% of the total entries, including missing parameters, swapped twist and rise values, incorrect sign of twist angles, partial symmetries and bona fide errors. Our validation code, workflow and the validated parameters are publicly available.
CRYO-EM; VALIDATION; HELICAL SYMMETRY; ELECTRON MICROSCOPY DATA BANK
Off-target structural insights: ArnA and AcrB in bacterial membrane-protein cryo-EM analysis
Membrane-protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC and the regulatory complex HflKC. These systems maintain proteostasis by facilitating membrane-protein insertion, folding and degradation. To gain structural insights into a putative complex formed by FtsH and YidC, we performed single-particle cryogenic electron microscopy on detergent-solubilized membrane samples, from which FtsH and YidC were purified using Ni–NTA affinity and size-exclusion chromatography. Although SDS–PAGE analysis indicated high purity of these proteins, cryo-EM data sets unexpectedly yielded high-resolution structures of ArnA and AcrB at 4.0 and 2.9 Å resolution, respectively. ArnA is a bifunctional enzyme involved in lipid A modification and polymyxin resistance, while AcrB is a multidrug efflux transporter of the AcrAB–TolC system. ArnA and AcrB, known Ni–NTA purification contaminants, were also consistently detected by mass spectrometry in Strep-Tactin affinity-purified samples, validating their presence independently of affinity-tag selection. ArnA, which is typically cytoplasmic, was consistently found in membrane-isolated samples, indicating an association with membrane components. Only 2D class averages corresponding to the cytoplasmic AAA+ domain of FtsH were observed; neither side views of full-length FtsH nor densities corresponding to an intact FtsH–YidC complex could be identified, due to the conformational flexibility of the FtsH complex and its transient interaction with YidC, which limited particle alignment and stable classification in cryo-EM data sets. Two-dimensional class averages revealed additional particles resembling GroEL and cytochrome bo3 oxidase. These results underscore the utility of cryo-EM in uncovering off-target yet structurally well defined complexes, which may reflect physiologically relevant interactions or purification biases during membrane-protein overexpression.
CRYO-EM; ARNA; ACRB; MEMBRANE-PROTEIN EXPRESSION; PROTEIN QUALITY CONTROL
Crystal structures of 40- and 71-substitution variants of hydroxynitrile lyase from rubber tree
Hydroxynitrile lyase from Hevea brasiliensis (HbHNL) and the esterase SABP2 from Nicotiana tabacum share the α/β-hydrolase fold, a Ser–His–Asp catalytic triad and 44% sequence identity, yet catalyze different reactions. Prior studies showed that three active-site substitutions in HbHNL conferred weak esterase activity. To investigate how regions beyond the active site influence catalytic efficiency and active-site geometry, we engineered HbHNL variants with increasing numbers of substitutions to match SABP2. Variant HNL16 has all amino acids within 6.5 Å of the active site identical to SABP2, HNL40 those within 10 Å and HNL71 those within 14 Å. HNL16 exhibited poor esterase activity, whereas both HNL40 and HNL71 showed efficient esterase catalysis, demonstrating that residues beyond the immediate active site are critical for functional conversion. X-ray structures of HNL40 and HNL71 reveal a progressive shift in backbone positions toward those of SABP2, with r.m.s.d. values of 0.51 Å (HNL40) and 0.41 Å (HNL71) over the Cα atoms, and even smaller r.m.s.d.s within the active-site region. Both HNL40 and HNL71 show a restored oxyanion hole and an additional tunnel connecting the active site to the protein surface. This work demonstrates the essential role of distant, indirectly acting residues to catalysis in α/β-hydrolase enzymes.
ENGINEERED PROTEINS; HYDROXYNITRILE LYASES; [ALPHA]/[BETA]-HYDROLASE FOLD; ESTERASES; SABP2; SALICYLIC ACID-BINDING PROTEIN 2
Understanding domain swapping in the c-Src SH3 domain through hinge-loop mutagenesis
The c-Src SH3 domain is one of the best-characterized modular domains from a biophysical and structural point of view. This SH3 domain displays noncanonical alternative folding, forming 3D domain-swapped oligomers and amyloid fibrils. These features make this small protein an ideal model for studying these phenomena. Residues in the regions that favour unfolding of the monomer and those in the hinge loop have been deeply studied in proteins undergoing 3D domain swapping. To study the role of these residues in the unfolding of the c-Src SH3 domain, we have constructed several chimeric proteins by interchanging residues in the RT and n-Src loops between the c-Src SH3 and Abl SH3 domains. The RT (the region between β1 and β2) and n-Src (the region between β2 and β3) loops create two sides of the shallow hydrophobic groove where proline-rich motif sequences bind to the SH3 domain. In addition to the structural information, we have performed a biophysical characterization of these chimeric constructs. The c-Src SH3 domain bearing the loops of the Abl SH3 shows minor changes in stability. Interestingly, these replacements do not prevent the formation of domain-swapped dimers. However, the interchange of one or two loops within the Abl SH3 domain produces a noticeable reduction in its stability but does not promote the formation of 3D domain-swapped oligomers. Thus, our results indicate that although the composition of the hinge loop is likely to play a role in the interchange of structural elements to form the intertwined dimers, it is not the sole driving force in their formation.
SH3 DOMAINS; 3D DOMAIN SWAPPING; HINGE LOOP; OPEN/SECONDARY INTERFACE; CLOSED/PRIMARY INTERFACE
A high-resolution data set of fatty acid-binding protein structures. I. Dynamics of FABP4 and ligand binding
Fatty acid-binding proteins (FABPs) are involved in the uptake and intracellular trafficking of fatty acids for metabolic and gene-regulatory purposes. FABPs are known to associate with membranes and also enter the nucleus. Using NMR and a human FABP4 (hFABP4) preparation completely free of endogenous ligands, we studied the influence of fatty acids and inhibitors on the conformational flexibility and bicelle/membrane association of this isoform. Binding of fatty acids and ligands rigidifies hFABP4, particularly at the portal region where ligands enter the binding site. Depending on the nature of the ligand, hFABP4 stays associated with bicelles via the portal region or segregates into solution, a prerequisite for nuclear import using a nonclassical nuclear localization signal. These results indicate that different ligands can lead to different biological outcomes. One of the major determinants for FABP4 segregation is Phe58, which in X-ray crystal structures adopts different conformations as a function of ligand volume. It is possible that other FABP isoforms use a similar mechanism for ligand-dependent membrane detachment and activation of nuclear import.
NMR DYNAMICS; MICELLE AND BICELLE BINDING; RESIDUAL LIGANDS; FATTY ACID-BINDING PROTEINS
A high-resolution data set of fatty acid-binding protein structures. III. Unexpectedly high occurrence of wrong ligands
FABP4 has been implicated as a therapeutic target for treating diabetes and atherosclerosis. Structure-based drug design (SBDD) based on initial hits from high-throughput and fragment screens yielded 216 ligand-bound structures of human FABP3, FABP4 and FABP5 isoforms, many of which were at resolutions of better than 1.2 Å. An estimated 15% of the ligands had a different chemical composition to that expected from the starting materials or the final synthesis product, highlighting a potential problem inherent to all SBDD campaigns conducted at lower resolution. Apart from possible human error during compound registration, side reactions such as additions, eliminations, isomerizations, cyclizations and dimerizations were found that led to compounds capable of binding to FABP.
STRUCTURE-BASED DRUG DESIGN; FATTY ACID-BINDING PROTEINS; LIGAND BINDING; UNEXPECTED CHEMICAL COMPOSITION
Q-score as a reliability measure for protein, nucleic acid and small-molecule atomic coordinate models derived from 3DEM maps
Atomic coordinate models are important for the interpretation of 3D maps produced with cryoEM and cryoET (3D electron microscopy; 3DEM). In addition to visual inspection of such maps and models, quantitative metrics can inform about the reliability of the atomic coordinates, in particular how well the model is supported by the experimentally determined 3DEM map. A recently introduced metric, Q-score, was shown to correlate well with the reported resolution of the map for well fitted models. Here, we present new statistical analyses of Q-score based on its application to ∼10 000 maps and models archived in the EMDB (Electron Microscopy Data Bank) and PDB (Protein Data Bank). Further, we introduce two new metrics based on Q-score to represent each map and model relative to all entries in the EMDB and those with similar resolution. We explore through illustrative examples of proteins, nucleic acids and small molecules how Q-scores can indicate whether the atomic coordinates are well fitted to 3DEM maps and also whether some parts of a map may be poorly resolved due to factors such as molecular flexibility, radiation damage and/or conformational heterogeneity. These examples and statistical analyses provide a basis for how Q-scores can be interpreted effectively in order to evaluate 3DEM maps and atomic coordinate models prior to publication and archiving.
CRYOEM; Q-SCORES; VALIDATION; B FACTORS; STRUCTURE
A high-resolution data set of fatty acid-binding protein structures. II. Crystallographic overview, ligand classes and binding pose
Fatty acid-binding protein isoforms 4 and 5 are potential diabetes and atherosclerosis targets. During a drug-design program aiming at dual isoform-specific FABP4/5 inhibitors with little or no affinity for FABP3, a set of crystal structures with a median resolution of 1.2 Å was generated. The chemical space of the ligands covers various series in which the carboxylate and aliphatic groups of the natural fatty-acid ligands have been replaced by other moieties. A summary of binding modes of the chemical series is also given with respect to how isoform specificity was achieved. Additionally, several bromine-containing ligands were identified that allowed SAD phasing, yielding an independent experimental confirmation of their chemical composition.
ISOFORM SPECIFICITY; CRYSTAL PATHOLOGIES; LIGAND BINDING; FORTUITOUS SAD PHASING
Probabilistic single-particle cryo-EM ab initio 3D reconstruction in SIMPLE
Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires ab initio 3D reconstruction of density volume(s) from 2D images (particles). This large-scale inverse problem requires the determination of many million degrees of freedom from extremely noisy experimental measurements. Here, we introduce a new approach to probabilistic multi-volume ab initio 3D reconstruction for simultaneous estimation of the relative particle 3D orientations and partitioning of the particles into groups with distinct structural states. To account for further structural variability within the discrete state groups, due to for example regional disorder, flexibility or partial occupancy of associating ligands, we introduce a new method for adaptive non-uniform regularization based on iterated conditional modes (ICMs). Our ICM regularization approach can be viewed as a spatially varying real-space prior that optimizes the connectivity of the reconstructed density map(s). Our method is designed to run in real time as the microscope collects the data, which puts significant constraints on algorithm scalability and flexibility with regard to how new particles are incorporated. We describe the probabilistic optimization and non-uniform regularization theory in detail. Finally, we provide numerous benchmarking examples, both on publicly available standard test data sets and on data sets acquired at our cryo-EM facility at the National Cancer Institute, National Institutes of Health. The implementation of our new multi-volume ab initio 3D reconstruction approach is part of the SIMPLE software suite, which is provided open source at https://github.com/hael/SIMPLE.
CRYO-EM; SINGLE PARTICLE; RECONSTRUCTION; PROBABILISTIC; HETEROGENEITY
Crystal structure of coagulation factor XII N-terminal domains 1–5
Factor XIIa (FXIIa) is generated from its zymogen factor XII (FXII) by contact with polyanions such as inorganic polyphosphates. FXIIa cleaves the substrates prekallikrein and factor XI, triggering inflammatory cascades and plasma coagulation. From the N-terminus, FXII has fibronectin type II (FnII), epidermal growth factor-1 (EGF1), fibronectin type I (FnI), EGF2 and kringle domains. The N-terminal domains of FXII mediate polyanion and Zn2+ binding. To understand how ligand binding to polyanions and Zn2+ is coordinated across multiple domains, we determined the crystal structure of recombinant FXII domains 1–5 (FXIIHC5) to 3.4 Å resolution. A separate crystal structure of the isolated FXII FnII domain at 1.2 Å resolution revealed two bound Zn2+ ions. In FXIIHC5 a head-to-tail interaction is formed between the FnII and kringle domains, co-localizing the lysine-binding sites of the kringle domain and the cation-binding site of the FnII domain. Two FXIIHC5 monomers interlock, burying a large surface area of 2067 Å2, such that two kringle domains point outwards separated by a distance of 20 Å. The polyanion-binding site in the EGF1 domain is localized onto a plane together with the FnII and FnI domains. Using native mass spectrometry, we detected a major FXIIHC5 monomer peak and a minor dimer peak. Small-angle X-ray scattering and gel-filtration chromatography revealed the presence of monomers and dimers in solution. These FXII N-terminal domain structures provide a holistic framework to understand how the mosaic domain structure of FXII assembles diverse ligand-binding sites in three dimensions.
FACTOR XII; FIBRONECTIN TYPE II DOMAIN; KRINGLE DOMAIN; CRYSTAL STRUCTURE
Structural dynamics of IDR interactions in human SFPQ and implications for liquid–liquid phase separation
The proteins SFPQ (splicing factor proline- and glutamine-rich) and NONO (non-POU domain-containing octamer-binding protein) are members of the Drosophila behaviour/human splicing (DBHS) protein family, sharing 76% sequence identity in their conserved DBHS domain. These proteins are critical for elements of pre- and post-transcriptional regulation in mammals and are primarily located in paraspeckles: ribonucleoprotein bodies templated by NEAT1 long noncoding RNA. Regions that are structured and predicted to be disordered (IDRs) in DBHS proteins facilitate various interactions, including dimerization, polymerization, nucleic acid binding and liquid–liquid phase separation, all of which have consequences for cell health, the pathology of some neurological diseases and cancer. To date, very limited structural work has been carried out on characterizing the IDRs of the DBHS proteins, largely due to their predicted disordered nature and the fact that this is often a bottleneck for conventional structural techniques. This is a problem worth addressing, as the IDRs have been shown to be critical to the material state of the protein as well as its function. In this study, we used small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), together with lysine cross-linking mass spectrometry (XL-MS), to investigate the regions of SFPQ flanking the structured DBHS domain and the possibility of dimer partner exchange of full-length proteins. Our results demonstrate experimentally that the N- and C-terminal regions on either side of the folded DBHS domain are long, disordered and flexible in solution. Realistic modelling of disordered chains to fit the scattering data and the compaction of the different protein variants suggests that it is physically possible for the IDRs to be close enough to interact. The mass-spectrometry data additionally indicate that the C-terminal IDR can potentially interact with the folded DBHS domain and also shares some conformational space with the N-terminal IDR. Our small-angle neutron scattering (SANS) experiments reveal that full-length SFPQ is capable of swapping dimer partners with itself, which has implications for our understanding of the combinatorial dimerization of DBHS proteins within cells. Our study provides insight into possible interactions between different IDRs either in cis or in trans and how these may relate to protein function, and the possible impact of mutations in these regions. The dynamic dimer partner exchange of a full-length protein inferred from this study is a phenomenon that is integral to the function of DBHS proteins, allowing changes in gene-regulatory activity by altering levels of the various heterodimers or homodimers.
PHASE SEPARATION; DISORDER; FLEXIBILITY; DIMERS; DBHS
Enhanced capabilities for multi-crystal data collection based on double mesh scans
Two-dimensional X-ray mesh (raster) scans are broadly used in macromolecular crystallography to identify positions on the sample holder at which diffraction images can be collected. Based on a single mesh scan, the commonly known method Mesh&Collect allows the collection of small angular wedges of diffraction data from multiple crystals identified in the mesh-scan area. Recently, the capabilities of the method have been extended by using two mesh scans instead of one. The scans are usually performed at orthogonal orientations of the rotation axis, or any other angular difference less than 90° can be used. This allows the more accurate determination, when compared with a single mesh scan, of the 3D centre positions of each crystal contained in the scanned area and gives better estimates of their dimensions and diffraction qualities. We present the upgraded DoubleMesh&Collect method based on the Dozor, Dozor-m2 and Resheteau software programs which automatically analyse the diffraction images of the two mesh scans. The applicability of the method is demonstrated on several cases, including room-temperature diffraction studies. The problem of cartography of large crystals is discussed.
DOZOR; DOZOR-M2; RESHETEAU; MACROMOLECULAR CRYSTALLOGRAPHY; X-RAY MESH RASTER SCANS; SAMPLE LOCALIZATION; DATA-COLLECTION STRATEGIES
Enhanced intensity-based clustering of isomorphous multi-crystal data sets in the presence of subtle variations
Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.
MULTI-CRYSTAL DATA SETS; DATA PROCESSING; CLUSTERING; AUTOMATION
Myricetin-bound crystal structure of the SARS-CoV-2 helicase NSP13 facilitates the discovery of novel natural inhibitors
The SARS-CoV-2 helicase NSP13 is a highly conserved and essential component of the viral replication machinery, making it a promising target for antiviral drug development. Here, we present the 2 Å resolution crystal structure of NSP13 bound to the natural flavonoid myricetin, revealing a conserved allosteric binding site. Guided by these structural findings, a virtual screening campaign identified the caffeic acid derivatives rosmarinic acid and chlorogenic acid as potential novel natural inhibitors, which were experimentally validated to inhibit RNA-unwinding activity. This study provides structural insights that could support ongoing drug-discovery efforts targeting NSP13 in SARS-CoV-2 and other coronaviruses with pandemic potential.
SARS-COV-2; NSP13 HELICASE; MYRICETIN; VIRTUAL SCREENING; NATURAL INHIBITORS; CAFFEIC ACID DERIVATIVES; ANTIVIRAL DRUG DEVELOPMENT
Robust error calibration for serial crystallography
Serial crystallography is an important technique with unique abilities to resolve enzymatic transition states, minimize radiation damage to sensitive metallo­enzymes and perform de novo structure determination from micrometre-sized crystals. This technique requires the merging of data from thousands of crystals, making manual identification of errant crystals unfeasible. cctbx.xfel.merge uses filtering to remove problematic data. However, this process is imperfect, and data reduction must be robust to outliers. We add robustness to cctbx.xfel.merge at the step of uncertainty determination for reflection intensities. This step is a critical point for robustness because it is the first step where the data sets are considered as a whole, as opposed to individual lattices. Robustness is conferred by reformulating the error-calibration procedure to have fewer and less stringent statistical assumptions and incorporating the ability to down-weight low-quality lattices. We then apply this method to five macromolecular XFEL data sets and observe the improvements to each. The appropriateness of the intensity uncertainties is demonstrated through internal consistency. This is performed through theoretical CC1/2 and I/σ relationships and by weighted second moments, which use Wilson's prior to connect intensity uncertainties with their expected distribution. This work presents new mathematical tools to analyze intensity statistics and demonstrates their effectiveness through the often underappreciated process of uncertainty analysis.
UNCERTAINTY QUANTIFICATION; ERROR CALIBRATION; SERIAL CRYSTALLOGRAPHY; XFELS
Structures of α-galactosaminidases from the CAZy GH114 family and homologs defining a new GH191 family of glycosidases
Endo-galactosaminidases are an underexplored family of enzymes involved in the degradation of galactosaminogalactan (GAG) and other galactosamine-containing cationic exopolysaccharides produced by fungi and bacteria. These exopolysaccharides are part of the cell wall and extracellular matrix of microbial communities. Currently, these galactosaminidases are found in three distinct CAZy families: GH114, GH135 and GH166. Despite the widespread occurrence of these enzymes in nearly all bacterial and fungal clades, only limited biochemical and structural data are available for these three groups. To expand our knowledge of endo-galactosaminidases, we selected several sequences predicted to encode endo-galactosaminidases and produced them recombinantly for structural and functional studies. Only very few predicted proteins could be produced in soluble form, and activity against bacterial Pel (pellicle) polysaccharide could only be confirmed for one enzyme. Here, we report the structures of two bacterial and one fungal enzyme. Whereas the fungal enzyme belongs to family GH114, the two bacterial enzymes do not lie in the current GH families but instead define a new family, GH191. During structure solution we realized that crystals of all three enzymes had various defects including twinning and partial disorder, which in the case of a more severe pathology in one of the structures required the design of a specialized refinement/model-building protocol. Comparison of the structures revealed several features that might be responsible for the described activity pattern and substrate specificity compared with other GAG-degrading enzymes.
GALACTOSAMINIDASES; STRUCTURE; GLYCOSIDE HYDROLASE FAMILY; CRYSTAL PATHOLOGY
Structure of the Fab fragment of a humanized 5E5 antibody to a cancer-specific Tn-MUC1 epitope
The structure of the humanized Fab from murine monoclonal antibody 5E5 specific for tumor antigen Tn-MUC1 has been determined to 1.57 Å resolution. Despite undertaking thousands of crystallization trials of the humanized 5E5 (h-5E5) Fab in the presence of either the singly or doubly glycosylated peptide antigens corresponding to Tn-MUC1, the Fab is only observed unliganded in the crystal. The conformations of the complementarity-determining regions (CDRs) of the combining site on the h-5E5 Fab do not differ significantly from those reported for liganded murine scFv at 3.0 Å resolution. While the affinity of the murine 5E5 has previously been reported as KD = 1.7 nM for the 24-mer Tn-MUC1 peptide PPAHGVT*SAPDTRPAPGS*T*APPAH prepared by in vitro glycosylation of a synthetic 24-mer MUC1 peptide, the KD of the h-5E5 Fab for the shorter doubly glycosylated glycopeptide antigens PAPGS*T*AP and APGS*T*AP was measured here as only 41 and 61 µM, respectively. Interestingly, the single Fab molecule in the asymmetric unit of space group C2 is observed packed head-to-head with a symmetry-related Fab across a crystallo­graphic twofold axis such that a polypeptide loop from the light chain of each Fab is observed to insert into the antigen-binding pocket of the symmetry-related Fab. While this might suggest that binding of the Tn-MUC1 peptides may have been inhibited by a homophilic association, none was detected. The humanization process has imposed changes in the framework regions of the Fv which may have affected the Vh–Vl interface.
CANCER; ANTI-TN-MUC1 ANTIBODY; 5E5; TN ANTIGEN; X-RAY CRYSTALLOGRAPHY; HUMANIZED ANTIBODIES; FAB STRUCTURE
InstaMap: instant-NGP for cryo-EM density maps
Despite the parallels between problems in computer vision and cryo-electron microscopy (cryo-EM), many state-of-the-art approaches from computer vision have yet to be adapted for cryo-EM. Within the computer-vision research community, implicits such as neural radiance fields (NeRFs) have enabled the detailed reconstruction of 3D objects from few images at different camera-viewing angles. While other neural implicits, specifically density fields, have been used to map conformational heterogeneity from noisy cryo-EM projection images, most approaches represent volume with an implicit function in Fourier space, which has disadvantages compared with solving the problem in real space, complicating, for instance, masking, constraining physics or geometry, and assessing local resolution. In this work, we build on a recent development in neural implicits, a multi-resolution hash-encoding framework called instant-NGP, that we use to represent the scalar volume directly in real space and apply it to the cryo-EM density-map reconstruction problem (InstaMap). We demonstrate that for both synthetic and real data, InstaMap for homogeneous reconstruction achieves higher resolution at shorter training stages than five other real-spaced representations. We propose a solution to noise overfitting, demonstrate that InstaMap is both lightweight and fast to train, implement masking from a user-provided input mask and extend it to molecular-shape heterogeneity via bending space using a per-image vector field.
CRYO-EM; DENSITY MAPS; HETEROGENEITY; END-TO-END GRADIENT-BASED LEARNING
Duplicate entries in the Protein Data Bank: how to detect and handle them
A global analysis of protein crystal structures in the Protein Data Bank (PDB) using a newly developed computational approach reveals many pairs with (nearly) identical main-chain coordinates. Such cases are identified and analyzed, showing that duplication is possible since the PDB does not currently have tools or mechanisms that would detect potentially duplicate submissions. Some duplicated entries represent modeling efforts of ligand binding that masquerade as experimentally determined structures. We propose that duplicate entries should either be obsoleted by the PDB or, as a minimum, marked with a clear `CAVEAT' record that would alert potential users to the presence of such problems. We also suggest that using a tool for verifying the uniqueness of the deposited structure, such as that presented in this work, should become part of the routine validation procedure for new depositions.
DATA DUPLICATION; PROTEIN DATA BANK; COORDINATE COMPARISON; BACKBONE RIGID INVARIANT
Human dystrophin tandem calponin homology actin-binding domain crystallized in a closed-state conformation
The structure of the N-terminal actin-binding domain of human dystrophin was determined at 1.94 Å resolution. Each chain in the asymmetric unit exists in a `closed' conformation, with the first and second calponin homology (CH) domains directly interacting via a 2500.6 Å2 interface. The positioning of the individual CH domains is comparable to the domain-swapped dimer seen in previous human dystrophin and utrophin actin-binding domain 1 structures. The CH1 domain is highly similar to the actin-bound utrophin structure and structural homology suggests that the `closed' single-chain conformation opens during actin binding to mitigate steric clashes between CH2 and actin.
ACTIN BINDING; DYSTROPHIN; DMD; CYTOSKELETON; CALPONIN HOMOLOGY
Slice'N'Dice: maximizing the value of predicted models for structural biologists
With the advent of next-generation modelling methods, such as AlphaFold2, structural biologists are increasingly using predicted structures to obtain structure solutions via molecular replacement (MR) or model fitting in single-particle cryogenic sample electron microscopy (cryoEM). Differences between the domain–domain orientations represented in a predicted model and a crystal structure are often a key limitation when using predicted models. Slice'N'Dice is a software package designed to address this issue by first slicing models into distinct structural units and then automatically placing the slices using either Phaser, MOLREP or PowerFit. The slicing step can use the AlphaFold predicted aligned error (PAE) or can operate via a variety of Cα-atom-based clustering algorithms, extending the applicability to structures of any origin. The number of splits can either be selected by the user or determined automatically. Slice'N'Dice is available for both MR and automated map fitting in the CCP4 and CCP-EM software suites.
STRUCTURE DETERMINATION; CRYO-EM; X-RAY CRYSTALLOGRAPHY; MOLECULAR REPLACEMENT; STRUCTURE PREDICTION
Expansion of the diversity of dispersin scaffolds
Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-d-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering
ENZYME CATALYSIS; GLYCOSIDE HYDROLASES; POLY-N-ACETYLGLUCOSAMINE; PROTEIN CRYSTALLOGRAPHY; GH20