DeepTracer: Fast Cryo-EM Protein Structure Modeling and Special Studies on CoV-related Complexes

Pfab, Jonas; Nm, Phan; D, Si

doi:10.1101/2020.07.21.214064

Cited by 9 publications

(7 citation statements)

References 34 publications

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“…Processing in cryoSPARC2 17,18 followed by RELION3 19 yielded a reconstruction of Nsp2 at 3.8 Å global resolution ranging from 3Å in the best resolved regions to 6Å in the most flexible regions (Fig 1A and Sup Fig 2). The initial model was automatically built into the well-resolved region using the DeepTracer 20,21 online server and then further corrected and refined via manual manipulation in Coot 22 and ISOLDE 23 , followed by final refinements in Phenix 24 and Rosetta 25 (see methods).…”

Section: Combining a Cryo-em Map With Alphafold2 Nsp2 Predictions Yields A Pseudo-atomic Structure For Full-length Nsp2mentioning

confidence: 99%

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes

Gupta¹,

Azumaya²,

Moritz³

et al. 2021

Preprint

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The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

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Section: Combining a Cryo-em Map With Alphafold2 Nsp2 Predictions Yields A Pseudo-atomic Structure For Full-length Nsp2mentioning

confidence: 99%

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes

Gupta¹,

Azumaya²,

Moritz³

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…Only the Ubr-Box1 structure was determined previously 24 . To overcome the difficulty during model building, we used the artificial intelligence (AI)-based do novo modelling tool DeepTracer 25 to build a starting model of the entire complex (see Methods for details) and manually adjusted and refined the model using COOT 26 . The final model was refined in real space using PHENIX 27 (Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…Only was the structure of Ubr-Box1 domain determined previously 24 . To overcome the difficulty during model building, we used artificial intelligence (AI) based de novo modelling tool DeepTracer program 25 to build a starting model of the entire complex from scratch. Specifically, the sharpened map of the initiation complex and a FASTA file containing sequences of Ubr1, Ubc2, and Ub were input to the online server of DeepTracer (https://deeptracer.uw.edu/home).…”

Section: Model Building Refinement and Validationmentioning

confidence: 99%

Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

Pan¹,

Zheng

Wang

et al. 2021

Preprint

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The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalyzed by Ubr1.

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“…Improvements in cryo-EM data collection and processing techniques in recent years have enabled structure determination at near-atomic resolutions ( Subramaniam, 2019 ). For structure interpretation, a number of tools for ab-initio model building have been developed and used in recent years ( Hoh et al, 2020 ; Terwilliger et al, 2020 ; Pfab et al, 2021 ; Lawson et al, 2021 ). Despite the resolution revolution, the majority of maps (92%) deposited in the EM Data Bank ( https://www.ebi.ac.uk/pdbe/emdb/ ) are at resolutions worse than 3 Å, and the average resolution of maps this year is around 5 Å ( https://www.ebi.ac.uk/pdbe/emdb/statistics_sp_res.html/ ).…”

Section: Introductionmentioning

confidence: 99%

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

Olek

Joseph

2021

Front. Mol. Biosci.

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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.

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DeepTracer: Fast Cryo-EM Protein Structure Modeling and Special Studies on CoV-related Complexes

Cited by 9 publications

References 34 publications

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes

Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

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

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