Combining Cryo-EM Density Map and Residue Contact for Protein Secondary Structure Topologies

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

Combine Cryo-EM Density Map and Residue Contact for Protein Structure Prediction: A Case Study

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 in 1D and a set of traces of secondary structures in 3D. In order to enhance the accuracy in ranking secondary structure topologies, we propose 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 seven cases show that a small set of secondary structure topologies can be produced to include the true topology when the three sources of information are used, even when errors exist in one or more of the three sources of information. The use of amino acid contact information improves the ranking of the true topology in six of the seven cases in the test.https://digitalcommons.odu.edu/gradposters2021_sciences/1004/thumbnail.jp

Analysis of Ab Initio Protein Structure Prediction Methods

Protein structure prediction produces atomic models of three-dimensional structure of a protein from its amino acid sequence. Understanding the function mechanism of proteins requires knowledge of three-dimensional structures. When developing new enzymes and drugs, it\u27s essential to understand the structure of the target protein. In this study, we analyze models predicted using two ab initio protein structure prediction methods, trRosetta and Quark. A set of thirty protein chains was used to evaluate the effectiveness of the methods. The thirty chains were collected from Protein Data Bank (June – November, 2020). The length and the relative position of the predicted secondary structures were examined. We found that the accuracy of models obtained from trRosetta and Quark is good (TM score 0.358 - 0.969). However, in some cases, the methods were not able to accurately predict the relative location of the secondary structures which might affect the overall folding relationship among secondary structures.https://digitalcommons.odu.edu/gradposters2023_sciences/1004/thumbnail.jp

Analysis of an Existing Method in Refinement of Protein Structure Predictions using Cryo-EM Images

Protein structure prediction produces atomic models from its amino acid sequence. Three-dimensional structures are important for understanding the function mechanism of proteins. Knowing the structure of a given protein is crucial in drug development design of novel enzymes. AlphaFold2 is a protein structure prediction tool with good performance in recent CASP competitions. Phenix is a tool for determination of a protein structure from a high-resolution 3D molecular image. Recent development of Phenix shows that it is capable to refine predicted models from AlphaFold2, specifically the poorly predicted regions, by incorporating information from the 3D image of the protein. The goal of this project is to understand the strengths and weaknesses of the approach that combines Phenix and AlphaFold2 using broader data. This analysis may provide insights for enhancement of the approach.https://digitalcommons.odu.edu/gradposters2022_sciences/1000/thumbnail.jp

Refinement of AlphaFold2 Models Against Experimental and Hybrid 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 highresolution cryo-EM maps might sometimes trap AlphaFold2 models in local optima

Cylindrical Similarity Measurement for Helices in Medium-Resolution Cryo-Electron Microscopy Density Maps

Cryo-electron microscopy (cryo-EM) density maps at medium resolution (5-10 Å) reveal secondary structural features such as α-helices and β-sheets, but they lack the side chains details that would enable a direct structure determination. Among the more than 800 entries in the Electron Microscopy Data Bank (EMDB) of medium-resolution density maps that are associated with atomic models, a wide variety of similarities can be observed between maps and models. To validate such atomic models and to classify structural features, a local similarity criterion, the F1 score, is proposed and evaluated in this study. The F1 score is theoretically normalized to a range from zero to one, providing a local measure of cylindrical agreement between the density and atomic model of a helix. A systematic scan of 30,994 helices (among 3,247 protein chains modeled into medium-resolution density maps) reveals an actual range of observed F1 scores from 0.171 to 0.848, suggesting that the local similarity is quantified and discriminated as intended. The best (highest) F1 scores tend to be associated with regions that exhibit high and spatially homogeneous local resolution (between 5 Å to 7.5 Å) in the helical density. The proposed F1 scores can be used as a discriminative classifier for validation studies and as a ranking criterion for cryo-EM density features in databases.https://digitalcommons.odu.edu/gradposters2020_sciences/1001/thumbnail.jp

A Tool for Segmentation of Secondary Structures in 3D Cryo-EM Density Map Components Using Deep Convolutional Neural Networks

Although cryo-electron microscopy (cryo-EM) has been successfully used to derive atomic structures for many proteins, it is still challenging to derive atomic structures when the resolution of cryo-EM density maps is in the medium resolution range, such as 5–10 Å. Detection of protein secondary structures, such as helices and β-sheets, from cryo-EM density maps provides constraints for deriving atomic structures from such maps. As more deep learning methodologies are being developed for solving various molecular problems, effective tools are needed for users to access them. We have developed an effective software bundle, DeepSSETracer, for the detection of protein secondary structure from cryo-EM component maps in medium resolution. The bundle contains the network architecture and a U-Net model trained with a curriculum and gradient of episodic memory (GEM). The bundle integrates the deep neural network with the visualization capacity provided in ChimeraX. Using a Linux server that is remotely accessed by Windows users, it takes about 6 s on one CPU and one GPU for the trained deep neural network to detect secondary structures in a cryo-EM component map containing 446 amino acids. A test using 28 chain components of cryo-EM maps shows overall residue-level F1 scores of 0.72 and 0.65 to detect helices and β-sheets, respectively. Although deep learning applications are built on software frameworks, such as PyTorch and Tensorflow, our pioneer work here shows that integration of deep learning applications with ChimeraX is a promising and effective approach. Our experiments show that the F1 score measured at the residue level is an effective evaluation of secondary structure detection for individual classes. The test using 28 cryo-EM component maps shows that DeepSSETracer detects β-sheets more accurately than Emap2sec+, with a weighted average residue-level F1 score of 0.65 and 0.42, respectively. It also shows that Emap2sec+ detects helices more accurately than DeepSSETracer with a weighted average residue-level F1 score of 0.77 and 0.72 respectively

The Saudi Critical Care Society practice guidelines on the management of COVID-19 in the ICU: Therapy section

BACKGROUND: The rapid increase in coronavirus disease 2019 (COVID-19) cases during the subsequent waves in Saudi Arabia and other countries prompted the Saudi Critical Care Society (SCCS) to put together a panel of experts to issue evidence-based recommendations for the management of COVID-19 in the intensive care unit (ICU). METHODS: The SCCS COVID-19 panel included 51 experts with expertise in critical care, respirology, infectious disease, epidemiology, emergency medicine, clinical pharmacy, nursing, respiratory therapy, methodology, and health policy. All members completed an electronic conflict of interest disclosure form. The panel addressed 9 questions that are related to the therapy of COVID-19 in the ICU. We identified relevant systematic reviews and clinical trials, then used the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach as well as the evidence-to-decision framework (EtD) to assess the quality of evidence and generate recommendations. RESULTS: The SCCS COVID-19 panel issued 12 recommendations on pharmacotherapeutic interventions (immunomodulators, antiviral agents, and anticoagulants) for severe and critical COVID-19, of which 3 were strong recommendations and 9 were weak recommendations. CONCLUSION: The SCCS COVID-19 panel used the GRADE approach to formulate recommendations on therapy for COVID-19 in the ICU. The EtD framework allows adaptation of these recommendations in different contexts. The SCCS guideline committee will update recommendations as new evidence becomes available

Combining Cryo-EM Density Map and Residue Contact for Protein Secondary Structure Topologies

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

AlphaFold2 Model Refinement Using Structure Decoys

AlphaFold2-predicted protein structures inform many modeling techniques in structural biology, including the interpretation of cryogenic electron microscopy (cryo-EM) maps. However, the accuracy of the AlphaFold2 prediction, the quality of the experimental cryo-EM data, and the reliability of the model\u27s alignment with density may affect the accuracy of the interpretation. In this work, we explored a new refinement approach by generating unbiased structural decoys from the AlphaFold2 model via 3DRobot or elastic network model (ENM)-based ModeHunter. Our hope was that some of the decoys would resemble the true structure, and consequently, that the refinement problem could then be reduced to selecting the correct decoy from the decoy set that most closely resembles the experimental cryo-EM map. We explored a map/model pair from the structure of a lipid-preserved respiratory supercomplex, where AlphaFold2 previously struggled (TM-score: 0.52). In this specific case, we observed that the inherent bias of 3DRobot toward compact decoys limited the AlphaFold2 model enhancement (best decoy TM-score: 0.53), whereas ENM is capable of producing extended decoys that significantly improve the accuracy of the AlphaFold2 model (best decoy TM-score: 0.68)