Fitting Atomic Structures into Cryo-EM Maps by Coupling Deep Learning-Enhanced Map Processing with Global-Local Optimization.

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.

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 central to the study of large-scale molecular interactions and has produced three-dimensional (3D) density maps at various resolutions. Secondary structure element (SSE) identification from volumetric protein density maps is critical for de novo backbone structure derivation in cryo-EM. Multiple methods have been developed to detect helix and β-sheet from density maps at medium resolutions (~5-10A). β-barrel as a special β-sheet structure has been found in many proteins. However, currently there are no methods aimed to automatically and accurately extract the β-barrel as a complete chunk of density from cryo-EM map of β-barrel protein. We present an effective approach, BarrelMiner, to automatically extract the β-barrel region from cryo-EM density maps based on the conspicuous cylindrical shape of β-barrel. BarrelMiner has been tested using ten simulated density maps at 10A resolution and seven experimental cryo-EM maps between 5.5A and 8.25A resolution. The result suggests that BarrelMiner can be used for the detection of β-barrel from medium resolution cryo-EM density maps of β-barrel proteins.

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…

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/.

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.

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.

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.

Structural modeling of proteins from cryo-electron microscopy (cryo-EM) density maps is one of the challenging issues in structural biology. De novo modeling combined with flexible fitting refinement (FFR) has been widely used to build a structure of new proteins. In de novo prediction, artificial conformations containing local structural errors such as chirality errors, cis peptide bonds, and ring penetrations are frequently generated and cannot be easily removed in the subsequent FFR. Moreover, refinement can be significantly suppressed due to the low mobility of atoms inside the protein. To overcome these problems, we propose an efficient scheme for FFR, in which the local structural errors are fixed first, followed by FFR using an iterative simulated annealing (SA) molecular dynamics protocol with the united atom (UA) model in an implicit solvent model; we call this scheme “SAUA-FFR”. The best model is selected from multiple flexible fitting runs with various biasing force constants to reduce overfitting. We apply our scheme to the decoys obtained from MAINMAST and demonstrate an improvement of the best model of eight selected proteins in terms of the root-mean-square deviation, MolProbity score, and RWplus score compared to the original scheme of MAINMAST. Fixing the local structural errors can enhance the formation of secondary structures, and the UA model enables progressive refinement compared to the all-atom model owing to its high mobility in the implicit solvent. The SAUA-FFR scheme realizes efficient and accurate protein structure modeling from medium-resolution maps with less overfitting.

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.

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.

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.

Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography