Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2
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Abstract
Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry ( in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.
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SciScore for 10.1101/2021.02.04.429751: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell culture and transfection: Human embryonic kidney cells 293 (HEK293, ATCC) were cultured (DMEM High glucose 10% FBS) at 37°C, 5% CO2, and high humidity. HEK293suggested: NoneSoftware and Algorithms Sentences Resources Label-free quantification (LFQ) was performed with MaxQuant 1.551 using the default parameters. MaxQuantsuggested: (MaxQuant, RRID:SCR_014485)Identification of cross-links: The RAW data files from the mass spectrometer were converted to MGF format by Proteome Discoverer (Thermo). Proteome Discoverersuggested: (Proteome Discoverer, RRID:SCR_014477)PatchDock employs an … SciScore for 10.1101/2021.02.04.429751: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell culture and transfection: Human embryonic kidney cells 293 (HEK293, ATCC) were cultured (DMEM High glucose 10% FBS) at 37°C, 5% CO2, and high humidity. HEK293suggested: NoneSoftware and Algorithms Sentences Resources Label-free quantification (LFQ) was performed with MaxQuant 1.551 using the default parameters. MaxQuantsuggested: (MaxQuant, RRID:SCR_014485)Identification of cross-links: The RAW data files from the mass spectrometer were converted to MGF format by Proteome Discoverer (Thermo). Proteome Discoverersuggested: (Proteome Discoverer, RRID:SCR_014477)PatchDock employs an efficient rigid docking algorithm that maximizes geometric shape complementarity. PatchDocksuggested: (PatchDock, RRID:SCR_017589)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 10. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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