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- PDB-6xrz: The 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA... -

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Entry
Database: PDB / ID: 6xrz
TitleThe 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
ComponentsFrameshift Stimulation Element from the SARS-CoV-2 RNA Genome
KeywordsRNA / Frameshift Stimulation Element / SARS-CoV-2 / COVID-19
Function / homology: / RNA / RNA (> 10)
Function and homology information
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsZhang, K. / Zheludev, I. / Hagey, R. / Wu, M. / Haslecker, R. / Hou, Y. / Kretsch, R. / Pintilie, G. / Rangan, R. / Kladwang, W. ...Zhang, K. / Zheludev, I. / Hagey, R. / Wu, M. / Haslecker, R. / Hou, Y. / Kretsch, R. / Pintilie, G. / Rangan, R. / Kladwang, W. / Li, S. / Pham, E. / Souibgui, C. / Baric, R. / Sheahan, T. / Souza, V. / Glenn, J. / Chiu, W. / Das, R.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01AI148382 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129564 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01AI120943 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122579 United States
CitationJournal: bioRxiv / Year: 2020
Title: Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome.
Authors: Kaiming Zhang / Ivan N Zheludev / Rachel J Hagey / Marie Teng-Pei Wu / Raphael Haslecker / Yixuan J Hou / Rachael Kretsch / Grigore D Pintilie / Ramya Rangan / Wipapat Kladwang / Shanshan Li ...Authors: Kaiming Zhang / Ivan N Zheludev / Rachel J Hagey / Marie Teng-Pei Wu / Raphael Haslecker / Yixuan J Hou / Rachael Kretsch / Grigore D Pintilie / Ramya Rangan / Wipapat Kladwang / Shanshan Li / Edward A Pham / Claire Bernardin-Souibgui / Ralph S Baric / Timothy P Sheahan / Victoria D Souza / Jeffrey S Glenn / Wah Chiu / Rhiju Das
Abstract: Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome . The frameshift stimulation element (FSE) of the SARS-CoV- ...Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome . The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides . To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.
History
DepositionJul 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome


Theoretical massNumber of molelcules
Total (without water)28,2861
Polymers28,2861
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area15320 Å2
Number of models10

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Components

#1: RNA chain Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome


Mass: 28285.660 Da / Num. of mol.: 1 / Fragment: GB residues 13434-13521 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2
References: GenBank: 1860584649

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.028 MDa / Experimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Buffer solutionpH: 8
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 8.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 10222
Image scansMovie frames/image: 30

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Processing

EM software
IDNameVersionCategory
2EPU2image acquisition
4CTFFIND4.13CTF correction
7UCSF Chimera1.13.1model fitting
11RELION3.0.2classification
12RELION3.0.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109137 / Symmetry type: POINT

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