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)
S10OD021600
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)
U24GM129564
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)
R35GM122579
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P01AI120943
United States
Citation
Journal: 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
Deposition
Jul 14, 2020
-
Header (metadata) release
Aug 19, 2020
-
Map release
Aug 19, 2020
-
Update
Aug 19, 2020
-
Current status
Aug 19, 2020
Processing site: RCSB / Status: Released
-
Structure visualization
Movie
Surface view with section colored by density value
Entire : Frameshift Stimulation Element tagged by ATP-TTR3
Entire
Name: Frameshift Stimulation Element tagged by ATP-TTR3
Components
Complex: Frameshift Stimulation Element tagged by ATP-TTR3
-
Supramolecule #1: Frameshift Stimulation Element tagged by ATP-TTR3
Supramolecule
Name: Frameshift Stimulation Element tagged by ATP-TTR3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)
Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weight
Theoretical: 60 KDa
-
Experimental details
-
Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
0.15 mg/mL
Buffer
pH: 8
Grid
Details: unspecified
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 12380 / Average exposure time: 6.0 sec. / Average electron dose: 8.3 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
In the structure databanks used in Yorodumi, some data are registered as the other names, "COVID-19 virus" and "2019-nCoV". Here are the details of the virus and the list of structure data.
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)
EMDB accession codes are about to change! (news from PDBe EMDB page)
The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
The EM Navigator/Yorodumi systems omit the EMD- prefix.
Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator
Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.
Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi