Overview and applications of map and model validation tools in the CCP-EM software suite.

Cryogenic electron microscopy (cryo-EM) has recently been established as a powerful technique for solving macromolecular structures. Although the best resolutions achievable are improving, a significant majority of data are still resolved at resolutions worse than 3 Å, where it is non-trivial to build or fit atomic models. The map reconstructions and atomic models derived from the maps are also prone to errors accumulated through the different stages of data processing. Here, we highlight the need to evaluate both model geometry and fit to data at different resolutions. Assessment of cryo-EM structures from SARS-CoV-2 highlights a bias towards optimising the model geometry to agree with the most common conformations, compared to the agreement with data. We present the CoVal web service which provides multiple validation metrics to reflect the quality of atomic models derived from cryo-EM data of structures from SARS-CoV-2. We demonstrate that further refinement can lead to improvement of the agreement with data without the loss of geometric quality. We also discuss the recent CCP-EM developments aimed at addressing some of the current shortcomings.

Atomic model validation using the CCP-EM software suite.

Recently, there has been a dramatic improvement in the quality and quantity of data derived using cryogenic electron microscopy (cryo-EM). This is also associated with a large increase in the number of atomic models built. Although the best resolutions that are achievable are improving, often the local resolution is variable, and a significant majority of data are still resolved at resolutions worse than 3 Å. Model building and refinement is often challenging at these resolutions, and hence atomic model validation becomes even more crucial to identify less reliable regions of the model. Here, a graphical user interface for atomic model validation, implemented in the CCP-EM software suite, is presented. It is aimed to develop this into a platform where users can access multiple complementary validation metrics that work across a range of resolutions and obtain a summary of evaluations. Based on the validation estimates from atomic models associated with cryo-EM structures from SARS-CoV-2, it was observed that models typically favor adopting the most common conformations over fitting the observations when compared with the model agreement with data. At low resolutions, the stereochemical quality may be favored over data fit, but care should be taken to ensure that the model agrees with the data in terms of resolvable features. It is demonstrated that further re-refinement can lead to improvement of the agreement with data without the loss of geometric quality. This also highlights the need for improved resolution-dependent weight optimization in model refinement and an effective test for overfitting that would help to guide the refinement process.

Cryo-EM Map-Based Model Validation Using the False Discovery Rate Approach.

Significant technological developments and increasing scientific interest in cryogenic electron microscopy (cryo-EM) has resulted in a rapid increase in the amount of data generated by these experiments and the derived atomic models. Robust measures for the validation of 3D reconstructions and atomic models are essential for appropriate interpretation of the data. The resolution of data and availability of software tools that work across a range of resolutions often limit the quality of derived models. Hence, the final atomic model is often incomplete or contains regions where atomic positions are less reliable or incorrectly built. Extensive manual pruning and local adjustments or rebuilding are usually required to address these issues. The presented research introduces a software tool for the validation of the backbone trace of atomic models built in the cryo-EM density maps. In this study, we use the false discovery rate analysis, which can be used to segregate molecular signals from the background. Each atomic position in the model can be associated with an FDR backbone validation score, which can be used to identify potential mistraced residues. We demonstrate that the proposed validation score is complementary to existing validation metrics and is useful especially in cases where the model is built in the maps having varying local resolution. We also discuss the application of the score for automated pruning of atomic models built ab-initio during the iterative model building process in Buccaneer. We have implemented this score in the CCP-EM software suite.

Assessment of protein-protein interfaces in cryo-EM derived assemblies.

Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we comprehensively assessed protein-protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed Protein Interface-score (PI-score), a density-independent machine learning-based metric, trained using the features of protein-protein interfaces in crystal structures. We evaluated 5873 interfaces in 1053 PDB-deposited cryo-EM models (including SARS-CoV-2 complexes), as well as the models submitted to CASP13 cryo-EM targets and the EM model challenge. We further inspected the interfaces associated with low-scores and found that some of those, especially in intermediate-to-low resolution (worse than 4 Å) structures, were not captured by density-based assessment scores. A combined score incorporating PI-score and fit-to-density score showed discriminatory power, allowing our method to provide a powerful complementary assessment tool for the ever-increasing number of complexes solved by cryo-EM.

TopoStats - A program for automated tracing of biomolecules from AFM images.

We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, ~90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub (https://github.com/afm-spm/TopoStats) and is intended to be modified and adapted for use if required.

TEMPy2: a Python library with improved 3D electron microscopy density-fitting and validation workflows.

Structural determination of molecular complexes by cryo-EM requires large, often complex processing of the image data that are initially obtained. Here, TEMPy2, an update of the TEMPy package to process, optimize and assess cryo-EM maps and the structures fitted to them, is described. New optimization routines, comprehensive automated checks and workflows to perform these tasks are described.

Making the invisible enemy visible.

During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, 548 structures covering 16 of the SARS-CoV-2 viral proteins have been released in a span of only 6 months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination - and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others.

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparing Cryo-EM Reconstructions and Validating Atomic Model Fit Using Difference Maps.

Cryogenic electron microscopy (cryo-EM) is a powerful technique for determining structures of multiple conformational or compositional states of macromolecular assemblies involved in cellular processes. Recent technological developments have led to a leap in the resolution of many cryo-EM data sets, making atomic model building more common for data interpretation. We present a method for calculating differences between two cryo-EM maps or a map and a fitted atomic model. The proposed approach works by scaling the maps using amplitude matching in resolution shells. To account for variability in local resolution of cryo-EM data, we include a procedure for local amplitude scaling that enables appropriate scaling of local map contrast. The approach is implemented as a user-friendly tool in the CCP-EM software package. To obtain clean and interpretable differences, we propose a protocol involving steps to process the input maps and output differences. We demonstrate the utility of the method for identifying conformational and compositional differences including ligands. We also highlight the use of difference maps for evaluating atomic model fit in cryo-EM maps.

iPBAvizu: a PyMOL plugin for an efficient 3D protein structure superimposition approach.

BACKGROUND: Protein 3D structure is the support of its function. Comparison of 3D protein structures provides insight on their evolution and their functional specificities and can be done efficiently via protein structure superimposition analysis. Multiple approaches have been developed to perform such task and are often based on structural superimposition deduced from sequence alignment, which does not take into account structural features. Our methodology is based on the use of a Structural Alphabet (SA), i.e. a library of 3D local protein prototypes able to approximate protein backbone. The interest of a SA is to translate into 1D sequences into the 3D structures. RESULTS: We used Protein blocks (PB), a widely used SA consisting of 16 prototypes, each representing a conformation of the pentapeptide skeleton defined in terms of dihedral angles. Proteins are described using PB from which we have previously developed a sequence alignment procedure based on dynamic programming with a dedicated PB Substitution Matrix. We improved the procedure with a specific two-step search: (i) very similar regions are selected using very high weights and aligned, and (ii) the alignment is completed (if possible) with less stringent parameters. Our approach, iPBA, has shown to perform better than other available tools in benchmark tests. To facilitate the usage of iPBA, we designed and implemented iPBAvizu, a plugin for PyMOL that allows users to run iPBA in an easy way and analyse protein superimpositions. CONCLUSIONS: iPBAvizu is an implementation of iPBA within the well-known and widely used PyMOL software. iPBAvizu enables to generate iPBA alignments, create and interactively explore structural superimposition, and assess the quality of the protein alignments.

Cryo-electron microscopy targets in CASP13: Overview and evaluation of results.

Structures of seven CASP13 targets were determined using cryo-electron microscopy (cryo-EM) technique with resolution between 3.0 and 4.0 Å. We provide an overview of the experimentally derived structures and describe results of the numerical evaluation of the submitted models. The evaluation is carried out by comparing coordinates of models to those of reference structures (CASP-style evaluation), as well as checking goodness-of-fit of modeled structures to the cryo-EM density maps. The performance of contributing research groups in the CASP-style evaluation is measured in terms of backbone accuracy, all-atom local geometry and similarity of inter-subunit interfaces. The results on the cryo-EM targets are compared with those on the whole set of eighty CASP13 targets. A posteriori refinement of the best models in their corresponding cryo-EM density maps resulted in structures that are very close to the reference structure, including some regions with better fit to the density.

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers.

The membrane attack complex (MAC) is one of the immune system's first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant ß-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ß-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

PBxplore: a tool to analyze local protein structure and deformability with Protein Blocks.

This paper describes the development and application of a suite of tools, called PBxplore, to analyze the dynamics and deformability of protein structures using Protein Blocks (PBs). Proteins are highly dynamic macromolecules, and a classical way to analyze their inherent flexibility is to perform molecular dynamics simulations. The advantage of using small structural prototypes such as PBs is to give a good approximation of the local structure of the protein backbone. More importantly, by reducing the conformational complexity of protein structures, PBs allow analysis of local protein deformability which cannot be done with other methods and had been used efficiently in different applications. PBxplore is able to process large amounts of data such as those produced by molecular dynamics simulations. It produces frequencies, entropy and information logo outputs as text and graphics. PBxplore is available at https://github.com/pierrepo/PBxplore and is released under the open-source MIT license.

Structural basis of human kinesin-8 function and inhibition.

Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their function. Equally important, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality, with microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end-directed motility. The Kif18A motor domain also depolymerizes microtubule plus and minus ends. BTB-1 inhibits both of these microtubule-based Kif18A activities. A reconstruction of BTB-1-bound, microtubule-bound Kif18A, in combination with computational modeling, identified an allosteric BTB-1-binding site near loop5, where it blocks the ATP-dependent conformational changes that we characterized. Strikingly, BTB-1 binding is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small-molecule binding near loop5 prevents a range of conformational changes, blocking motor function.

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

Integrative modelling of cellular assemblies.

A wide variety of experimental techniques can be used for understanding the precise molecular mechanisms underlying the activities of cellular assemblies. The inherent limitations of a single experimental technique often requires integration of data from complementary approaches to gain sufficient insights into the assembly structure and function. Here, we review popular computational approaches for integrative modelling of cellular assemblies, including protein complexes and genomic assemblies. We provide recent examples of integrative models generated for such assemblies by different experimental techniques, especially including data from 3D electron microscopy (3D-EM) and chromosome conformation capture experiments, respectively. We highlight general concepts in integrative modelling and discuss the need for careful formulation and merging of different types of information.

Improved metrics for comparing structures of macromolecular assemblies determined by 3D electron-microscopy.

Recent developments in 3-dimensional electron microcopy (3D-EM) techniques and a concomitant drive to look at complex molecular structures, have led to a rapid increase in the amount of volume data available for biomolecules. This creates a demand for better methods to analyse the data, including improved scores for comparison, classification and integration of data at different resolutions. To this end, we developed and evaluated a set of scoring functions that compare 3D-EM volumes. To test our scores we used a benchmark set of volume alignments derived from the Electron Microscopy Data Bank. We find that the performance of different scores vary with the map-type, resolution and the extent of overlap between volumes. Importantly, adding the overlap information to the local scoring functions can significantly improve their precision and accuracy in a range of resolutions. A combined score involving the local mutual information and overlap (LMI_OV) performs best overall, irrespective of the map category, resolution or the extent of overlap, and we recommend this score for general use. The local mutual information score itself is found to be more discriminatory than cross-correlation coefficient for intermediate-to-low resolution maps or when the map size and density distribution differ significantly. For comparing map surfaces, we implemented two filters to detect the surface points, including one based on the 'extent of surface exposure'. We show that scores that compare surfaces are useful at low resolutions and for maps with evident surface features. All the scores discussed are implemented in TEMPy (http://tempy.ismb.lon.ac.uk/).