Efficient Flexible Fitting Refinement with Automatic Error Fixing for De Novo Structure Modeling from Cryo-EM Density Maps

The rigid-body fitting of predicted structural models into cryo-electron microscopy (cryo-EM) density maps is a necessary procedure for density map-guided protein structure determination and prediction. We proposed a novel multiobjective optimization protocol, MOFIT, which performs a rigid-body density-map fitting based on particle swarm optimization (PSO). MOFIT was tested on a large set of 292 nonhomologous single-domain proteins. Starting from structural models predicted by I-TASSER, MOFIT achieved an average coordinate root-mean-square deviation of 2.46 Å, which was 1.57, 2.79, and 3.95 Å lower than three leading single-objective function-based methods, where the differences were statistically significant with p-values of 1.65 × 10-6, 6.36 × 10-8, and 6.44 × 10-11 calculated using two-tail Student's t tests. Detailed analyses showed that the major advantages of MOFIT lie in the multiobjective protocol and the extensive PSO search simulations guided by the composite objective functions, which integrates complementary correlation coefficients from the global structure, local fragments, and individual residues with the cryo-EM density maps.

The two major challenges in protein structure prediction problems are (1) the lack of an accurate energy function and (2) the lack of an efficient search algorithm. A protein energy function accurately describing the interaction between residues is able to supervise the optimization of a protein conformation, as well as select native or native-like structures from numerous possible conformations. An efficient search algorithm must be able to reduce a conformational space to a reasonable size without missing the native conformation. My PhD research studies focused on these two directions. A protein energy function—the distance and orientation dependent energy function of amino acid key blocks (DOKB), containing a distance term, an orientation term, and a highly packed term—was proposed to evaluate the stability of proteins. In this energy function, key blocks of each amino acids were used to represent each residue; a novel reference state was used to normalize block distributions. The dependent relationship between the orientation term and the distance term was revealed, representing the preference of different orientations at different distances between key blocks. Compared with four widely used energy functions using six general benchmark decoy sets, the DOKB appeared to perform very well in recognizing native conformations. Additionally, the highly packed term in the DOKB played its important role in stabilizing protein structures containing highly packed residues. The cluster potential adjusted the reference state of highly packed areas and significantly improved the recognition of the native conformations in the ig_structal data set. The DOKB is not only an alternative protein energy function for protein structure prediction, but it also provides a different view of the interaction between residues. The top-k search algorithm was optimized to be used for proteins containing both α-helices and β-sheets. Secondary structure elements (SSEs) are visible in cryo-electron microscopy (cryo-EM) density maps. Combined with the SSEs predicted in a protein sequence, it is feasible to determine the topologies referring to the order and direction of the SSEs in the cryo-EM density map with respect to the SSEs in the protein sequence. Our group member Dr. Al Nasr proposed the top-k search algorithm, searching the top-k possible topologies for a target protein. It was the most effective algorithm so far. However, this algorithm only works well for pure a-helix proteins due to the complexity of the topologies of β-sheets. Based on the known protein structures in the Protein Data Bank (PDB), we noticed that some topologies in β-sheets had a high preference; on the contrary, some topologies never appeared. The preference of different topologies of β-sheets was introduced into the optimized top-k search algorithm to adjust the edge weight between nodes. Compared with the previous results, this optimization significantly improved the performance of the top-k algorithm in the proteins containing both α-helices and…

As more protein structure models have been determined from cryogenic electron microscopy (cryo-EM) density maps, establishing how to evaluate the model accuracy and how to correct models in cases where they contain errors is becoming crucial to ensure the quality of the structural models deposited in the public database, the PDB. Here, a new protocol is presented for evaluating a protein model built from a cryo-EM map and applying local structure refinement in the case where the model has potential errors. Firstly, model evaluation is performed using a deep-learning-based model-local map assessment score, DAQ, that has recently been developed. The subsequent local refinement is performed by a modified AlphaFold2 procedure, in which a trimmed template model and a trimmed multiple sequence alignment are provided as input to control which structure regions to refine while leaving other more confident regions of the model intact. A benchmark study showed that this protocol, DAQ-refine, consistently improves low-quality regions of the initial models. Among 18 refined models generated for an initial structure, DAQ shows a high correlation with model quality and can identify the best accurate model for most of the tested cases. The improvements obtained by DAQ-refine were on average larger than other existing methods.

Advances in cryoelectron microscopy (cryo-EM) have revolutionized the structural investigation of large macromolecular assemblies. In this review, we first provide a broad overview of modeling methods used for flexible fitting of molecular models into cryo-EM density maps. We give special attention to approaches rooted in molecular simulations-atomistic molecular dynamics and Monte Carlo. Concise descriptions of the methods are given along with discussion of their advantages, limitations, and most popular alternatives. We also describe recent extensions of the widely used molecular dynamics flexible fitting (MDFF) method and discuss how different model-building techniques could be incorporated into new hybrid modeling schemes and simulation workflows. Finally, we provide two illustrative examples of model-building and refinement strategies employing MDFF, cascade MDFF, and RosettaCM. These examples come from recent cryo-EM studies that elucidated transcription preinitiation complexes and shed light on the functional roles of these assemblies in gene expression and gene regulation.

SummaryThe identification of transmembrane helices in transmembrane proteins is crucial, not only to understand their mechanism of action but also to develop new therapies. While experimental data on the boundaries of membrane-embedded regions are sparse, this information is present in cryo-electron microscopy (cryo-EM) density maps and it has not been utilized yet for determining membrane regions. We developed a computational pipeline, where the inputs of a cryo-EM map, the corresponding atomistic structure, and the potential bilayer orientation determined by TMDET algorithm of a given protein result in an output defining the residues assigned to the bulk water phase, lipid interface and the lipid hydrophobic core. Based on this method, we built a database involving published cryo-EM protein structures and a server to be able to compute this data for newly obtained structures.Availability and implementationhttp://memblob.hegelab.org.Supplementary informationSupplementary data are available at Bioinformatics online.

Cryo-electron microscopy (cryo-EM) has become a leading approach for protein structure determination, but it remains challenging to accurately model atomic structures with cryo-EM density maps. We propose a hybrid method, CR-I-TASSER, which integrates deep neural-network learning with I-TASSER assembly simulations for automated cryo-EM structure determination. The method is benchmarked on 778 proteins with simulated and experimental density maps, where CR-I-TASSER constructs models with a correct fold (TM-score>0.5) for 643 targets that is 64% higher than the best of other de novo and refinement-based approaches on high-resolution data samples. Detailed data analyses showed that the major advantage of CR-I-TASSER lies in the deep-learning based position prediction, which significantly improves the threading template quality and therefore boosts the accuracy of final models through optimized fragment assembly simulations. These results demonstrate a new avenue to determine cryo-EM protein structures with high accuracy and robustness covering various target types and density-map resolutions.

Although atomic structures have been determined directly from cryo-EM density maps with high resolutions, current structure determination methods for medium resolution (5 to 10 Å) cryo-EM maps are limited by the availability of structure templates. Secondary structure traces are lines detected from a cryo-EM density map for α-helices and β-strands of a protein. A topology of secondary structures defines the mapping between a set of sequence segments and a set of traces of secondary structures in three-dimensional space. In order to enhance accuracy in ranking secondary structure topologies, we explored a method that combines three sources of information: a set of sequence segments in 1D, a set of amino acid contact pairs in 2D, and a set of traces in 3D at the secondary structure level. A test of fourteen cases shows that the accuracy of predicted secondary structures is critical for deriving topologies. The use of significant long-range contact pairs is most effective at enriching the rank of the maximum-match topology for proteins with a large number of secondary structures, if the secondary structure prediction is fairly accurate. It was observed that the enrichment depends on the quality of initial topology candidates in this approach. We provide detailed analysis in various cases to show the potential and challenge when combining three sources of information.

Atomic structure modeling is a crucial step in determining the structures of protein complexes using cryo-electron microscopy (cryo-EM). This work introduces DEMO-EMol, an improved server that integrates deep learning-based map segmentation and chain fitting to accurately assemble protein-nucleic acid (NA) complex structures from cryo-EM density maps. Starting from a density map and independently modeled chain structures, DEMO-EMol first segments protein and NA regions from the density map using deep learning. The overall complex is then assembled by fitting protein and NA chain models into their respective segmented maps, followed by domain-level fitting and optimization for protein chains. The output of DEMO-EMol includes the final assembled complex model along with overall and residue-level quality assessments. DEMO-EMol was evaluated on a comprehensive benchmark set of cryo-EM maps with resolutions ranging from 1.96 to 12.77 Å, and the results demonstrated its superior performance over the state-of-the-art methods for both protein-NA and protein-protein complex modeling. The DEMO-EMol web server is freely accessible at https://zhanggroup.org/DEMO-EMol/.

Cryo-EM experiments produce images of macromolecular assemblies that are combined to produce three-dimensional density maps. Typically, atomic models of the constituent molecules are fitted into these maps, followed by a density-guided refinement. We introduce TEMPy-ReFF, a method for atomic structure refinement in cryo-EM density maps. Our method represents atomic positions as components of a Gaussian mixture model, utilising their variances as B-factors, which are used to derive an ensemble description. Extensively tested on a substantial dataset of 229 cryo-EM maps from EMDB ranging in resolution from 2.1-4.9 Å with corresponding PDB and CERES atomic models, our results demonstrate that TEMPy-ReFF ensembles provide a superior representation of cryo-EM maps. On a single-model basis, it performs similarly to the CERES re-refinement protocol, although there are cases where it provides a better fit to the map. Furthermore, our method enables the creation of composite maps free of boundary artefacts. TEMPy-ReFF is useful for better interpretation of flexible structures, such as those involving RNA, DNA or ligands.

Recent breakthroughs in deep learning-based protein structure prediction show that it is possible to obtain highly accurate models for a wide range of difficult protein targets for which only the amino acid sequence is known. The availability of accurately predicted models from sequences can potentially revolutionise many modelling approaches in structural biology, including the interpretation of cryo-EM density maps. Although atomic structures can be readily solved from cryo-EM maps of better than 4 Å resolution, it is still challenging to determine accurate models from lower-resolution density maps. Here, we report on the benefits of models predicted by AlphaFold2 (the best-performing structure prediction method at CASP14) on cryo-EM refinement using the Phenix refinement suite for AlphaFold2 models. To study the robustness of model refinement at a lower resolution of interest, we introduced hybrid maps (i.e. experimental cryo-EM maps) filtered to lower resolutions by real-space convolution. The AlphaFold2 models were refined to attain good accuracies above 0.8 TM scores for 9 of the 13 cryo-EM maps. TM scores improved for AlphaFold2 models refined against all 13 cryo-EM maps of better than 4.5 Å resolution, 8 hybrid maps of 6 Å resolution, and 3 hybrid maps of 8 Å resolution. The results show that it is possible (at least with the Phenix protocol) to extend the refinement success below 4.5 Å resolution. We even found isolated cases in which resolution lowering was slightly beneficial for refinement, suggesting that high-resolution cryo-EM maps might sometimes trap AlphaFold2 models in local optima.

MOTIVATION: Single-particle cryo electron microscopy (cryoEM) typically produces density maps of macromolecular assemblies at intermediate to low resolution (approximately 5-30 A). By fitting high-resolution structures of assembly components into these maps, pseudo-atomic models can be obtained. Optimizing the quality-of-fit of all components simultaneously is challenging due to the large search space that makes the exhaustive search over all possible component configurations computationally unfeasible.RESULTS: We developed an efficient mathematical programming algorithm that simultaneously fits all component structures into an assembly density map. The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. In contrast to other point matching algorithms, our algorithm is able to match multiple point sets simultaneously and not only based on their geometrical equivalence, but also based on the similarity of the density in the immediate point neighborhood. In addition, we present an efficient refinement method based on the Iterative Closest Point registration algorithm. The integer quadratic programming method generates an assembly configuration in a few seconds. This efficiency allows the generation of an ensemble of candidate solutions that can be assessed by an independent scoring function. We benchmarked the method using simulated density maps of 11 protein assemblies at 20 A, and an experimental cryoEM map at 23.5 A resolution. Our method was able to generate assembly structures with root-mean-square errors <6.5 A, which have been further reduced to <1.8 A by the local refinement procedure.AVAILABILITY: The program is available upon request as a Matlab code package.SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Online. PMID: 20529915 Funding information This work was supported by: Medical Research Council, United Kingdom Grant ID: G0600084 Biotechnology and Biological Sciences Research Council, United Kingdom Grant ID: G0600084

Recent breakthroughs in deep learning-based protein structure prediction show that it is possible to obtain highly accurate models for a wide range of difficult protein targets for which only the amino acid sequence is known. The availability of accurately predicted models from sequences can potentially revolutionise many modelling approaches in structural biology, including the interpretation of cryo-EM density maps. Although atomic structures can be readily solved from cryo-EM maps of better than 4 Å resolution, it is still challenging to determine accurate models from lower-resolution density maps. Here, we report on the benefits of models predicted by AlphaFold2 (the best-performing structure prediction method at CASP14) on cryo-EM refinement using the Phenix refinement suite for AlphaFold2 models. To study the robustness of model refinement at a lower resolution of interest, we introduced hybrid maps (i.e. experimental cryo-EM maps) filtered to lower resolutions by real-space convolution. The AlphaFold2 models were refined to attain good accuracies above 0.8 TM scores for 9 of the 13 cryo-EM maps. TM scores improved for AlphaFold2 models refined against all 13 cryo-EM maps of better than 4.5 Å resolution, 8 hybrid maps of 6 Å resolution, and 3 hybrid maps of 8 Å resolution. The results show that it is possible (at least with the Phenix protocol) to extend the refinement success below 4.5 Å resolution. We even found isolated cases in which resolution lowering was slightly beneficial for refinement, suggesting that high-resolution cryo-EM maps might sometimes trap AlphaFold2 models in local optima.

Recent breakthroughs in deep learning-based protein structure prediction show that it is possible to obtain highly accurate models for a wide range of difficult protein targets for which only the amino acid sequence is known. The availability of accurately predicted models from sequences can potentially revolutionise many modelling approaches in structural biology, including the interpretation of cryo-EM density maps. Although atomic structures can be readily solved from cryo-EM maps of better than 4 Å resolution, it is still challenging to determine accurate models from lower-resolution density maps. Here, we report on the benefits of models predicted by AlphaFold2 (the best-performing structure prediction method at CASP14) on cryo-EM refinement using the Phenix refinement suite for AlphaFold2 models. To study the robustness of model refinement at a lower resolution of interest, we introduced hybrid maps (i.e. experimental cryo-EM maps) filtered to lower resolutions by real-space convolution. The AlphaFold2 models were refined to attain good accuracies above 0.8 TM scores for 9 of the 13 cryo-EM maps. TM scores improved for AlphaFold2 models refined against all 13 cryo-EM maps of better than 4.5 Å resolution, 8 hybrid maps of 6 Å resolution, and 3 hybrid maps of 8 Å resolution. The results show that it is possible (at least with the Phenix protocol) to extend the refinement success below 4.5 Å resolution. We even found isolated cases in which resolution lowering was slightly beneficial for refinement, suggesting that high-resolution cryo-EM maps might sometimes trap AlphaFold2 models in local optima.

Bluetongue virus (BTV) is transmitted by blood-feeding insects (Culicoides sp.) and causes hemorrhagic diseases in livestock. BTV is a nonenveloped, double-stranded RNA (dsRNA) virus with two capsids: a well-studied, stable core enclosing the dsRNA genome and a highly unstable, poorly studied coat responsible for host cell attachment and entry. Here, based on cryo-electron microscopy (cryoEM), we report a 7-A resolution structure of the infectious BTV virion, including the coat proteins. We show that unlike other dsRNA viruses, the VP2 attachment trimer has a triskelion shape composed of three tip domains branching from a central hub domain. We identify three putative sialic acid-binding pockets in the hub and present supporting biochemical data indicating sugar moiety binding is important for BTV infection. Despite being a nonenveloped virus, the putative VP5 membrane penetration trimer, located slightly inward of the VP2 attachment trimer, has a central coiled-coil alpha-helical bundle, similar to the fusion proteins of many enveloped viruses (e.g., HIV, herpesviruses, vesicular stomatitis virus, and influenza virus). Moreover, mapping of the amino acid sequence of VP5 to the secondary structural elements identified by cryoEM locates 15 amphipathic alpha-helical regions on the external surface of each VP5 trimer. The cryoEM density map also reveals few, weak interactions between the VP5 trimer and both the outer-coat VP2 trimer and the underlying core VP7 trimer, suggesting that the surface of VP5 could unfurl like an umbrella during penetration and shedding of the coat to release the transcriptionally active core particle.

More and more deep learning approaches have been proposed to segment secondary structures from cryo-electron density maps at medium resolution range (5-10Å). Although the deep learning approaches show great potential, only a few small experimental data sets have been used to test the approaches. There is limited understanding about potential factors, in data, that affect the performance of segmentation. We propose an approach to generate data sets with desired specifications in three potential factors - the protein sequence identity, structural contents, and data quality. The approach was implemented and has generated a test set and various training sets to study the effect of secondary structure content and data quality on the performance of DeepSSETracer, a deep learning method that segments regions of protein secondary structures from cryo-EM map components. Results show that various content levels in the secondary structure and data quality influence the performance of segmentation for DeepSSETracer.