Expression, crystallization and preliminary crystallographic study of the C-terminal half of nsp2 from SARS coronavirus

Li, Yuanyuan; Ren, Zhilin; Bao, Zehua; Ming, Zhenhua; Li, Xuemei

doi:10.1107/s1744309111017829

Cited by 3 publications

(1 citation statement)

References 18 publications

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“…To obtain a model consistent with the cross-link set, we divided the AlphaFold2 model into domains (residues 1 to 104, 105 to 132, 133 to 275, 276 to 345, and 512 to 638). One domain that was not covered by the initial AlphaFold2 model (residues 359 to 511) was modeled by homology to partial Nsp2 structure of the infectious bronchitis virus [Protein Data Bank (PDB) ID code 3ld1 ( 30 ), sequence identity 13%]. With the availability of the structures for the individual domains, the modeling task is converted into a domain assembly problem.…”

Section: Resultsmentioning

confidence: 99%

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

Slavin

Zamel

Zohar

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 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 interdomain 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 cryogenic electron microscopy 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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Section: Resultsmentioning

confidence: 99%

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

Slavin

Zamel

Zohar

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Atlas of coronavirus replicase structure

et al. 2014

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The international response to SARS-CoV has produced an outstanding number of protein structures in a very short time. This review summarizes the findings of functional and structural studies including those derived from cryoelectron microscopy, small angle X-ray scattering, NMR spectroscopy, and X-ray crystallography, and incorporates bioinformatics predictions where no structural data is available. Structures that shed light on the function and biological roles of the proteins in viral replication and pathogenesis are highlighted. The high percentage of novel protein folds identified among SARS-CoV proteins is discussed.

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Targetedin situcross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

Slavin

Zamel

Zohar

et al. 2021

Preprint

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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.

show abstract

Expression, crystallization and preliminary crystallographic study of the C-terminal half of nsp2 from SARS coronavirus

Cited by 3 publications

References 18 publications

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

Atlas of coronavirus replicase structure

Targetedin situcross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

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