Dataset Information

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

ABSTRACT: Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with key host proteins and processes.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

DISEASE(S): Coronavirus Infectious Disease,Disease Free

SUBMITTER: Mehdi Bouhaddou

LAB HEAD: Nevan J Krogan

PROVIDER: PXD025817 | Pride | 2022-01-20

REPOSITORIES: Pride

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

Gupta Meghna M Azumaya Caleigh M CM Moritz Michelle M Pourmal Sergei S Diallo Amy A Merz Gregory E GE Jang Gwendolyn G Bouhaddou Mehdi M Fossati Andrea A Brilot Axel F AF Diwanji Devan D Hernandez Evelyn E Herrera Nadia N Kratochvil Huong T HT Lam Victor L VL Li Fei F Li Yang Y Nguyen Henry C HC Nowotny Carlos C Owens Tristan W TW Peters Jessica K JK Rizo Alexandrea N AN Schulze-Gahmen Ursula U Smith Amber M AM Young Iris D ID Yu Zanlin Z Asarnow Daniel D Billesbølle Christian C Campbell Melody G MG Chen Jen J Chen Kuei-Ho KH Chio Un Seng US Dickinson Miles Sasha MS Doan Loan L Jin Mingliang M Kim Kate K Li Junrui J Li Yen-Li YL Linossi Edmond E Liu Yanxin Y Lo Megan M Lopez Jocelyne J Lopez Kyle E KE Mancino Adamo A Moss Frank R FR Paul Michael D MD Pawar Komal Ishwar KI Pelin Adrian A Pospiech Thomas H TH Puchades Cristina C Remesh Soumya Govinda SG Safari Maliheh M Schaefer Kaitlin K Sun Ming M Tabios Mariano C MC Thwin Aye C AC Titus Erron W EW Trenker Raphael R Tse Eric E Tsui Tsz Kin Martin TKM Wang Feng F Zhang Kaihua K Zhang Yang Y Zhao Jianhua J Zhou Fengbo F Zhou Yuan Y Zuliani-Alvarez Lorena L Agard David A DA Cheng Yifan Y Fraser James S JS Jura Natalia N Kortemme Tanja T Manglik Aashish A Southworth Daniel R DR Stroud Robert M RM Swaney Danielle L DL Krogan Nevan J NJ Frost Adam A Rosenberg Oren S OS Verba Kliment A KA

bioRxiv : the preprint server for biology 20210511

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-C ...[more]

PMID: 34013269

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Project description:There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected pa-tient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as RT-PCR are the gold standard, during the current pandemic the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers, and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detec-tion methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syn-cytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagno-sis of active infection and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of pro-teins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein we report a tar-geted mass spectrometry assay for the detection of SARS- CoV-2 spike protein and nucleoprotein in a relevant biologi-cal matrix. Recombinant full-length spike protein and nucleoprotein were digested and prototypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high resolution Orbitrap instrument. A spectral library, which con-tained 7 proteotypic peptides (4 from spike protein and 3 from nucleoprotein) and the top 3 to 4 transitionsMS2 spectra, was generated and evaluated. From the original spectral library, we selected 2 best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in-vitro de-rived mucus. The PRM assay provided a limit of detection (LOD) of ~200 attomoles and a limit of quantitation (LOQ) of ~ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2E5 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the pres-ence of SARS-CoV-2 in archived or recently collected biological fluids, in-vitro derived research materials, and wastewater samples

			Action	DRS
	GFP.fasta	Fasta
	NSP2_PRM.blib	Other
	NSP2_PRM.redundant.blib	Other
	NSP2_PRM.sky	Other
	NSP2_PRM.sky.view	Other