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1.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-318315

ABSTRACT

Recent advances in single particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well-suited for routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.Funding: This work is supported by the Pels Foundation to The Rockefeller University, NIH grants P41 GM109824 and P41 GM103314 to BTC, R35 GM118130 to SAD, and R01 GM114450 to EAC. Access to the cryo-EM microscopes and support was through The Rockefeller University Evelyn Gruss Lipper Cryo-EM Resource Center and at The Simons Electron Microscopy Center (SEMC), National Resource for Automated Molecular Microscopy (NRAMM), and National Center for CryoEM Access and Training (NCCAT) at the NYSBC, supported by NIH NIGMS (P41 GM103310), NYSTAR, the Simons Foundation (SF349247), the NIH Common Fund Transformative High Resolution CryoElectron Microscopy program (U24 GM129539) and NY State Assembly Majority. Conflict of Interest: The authors declare there are no competing interests.

2.
Structure ; 29(2): 186-195.e6, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-939287

ABSTRACT

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.


Subject(s)
Cryoelectron Microscopy/methods , Mass Spectrometry/methods , Single Molecule Imaging/methods , Escherichia coli , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Methyltransferases/chemistry , Methyltransferases/metabolism , RNA Helicases/chemistry , RNA Helicases/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure , Transcription Factors/chemistry , Transcription Factors/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
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