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- EMDB-22296: The 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA... -

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Entry
Database: EMDB / ID: EMD-22296
TitleThe 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
Map data28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
Sample
  • Complex: 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
    • RNA: Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
KeywordsFrameshift Stimulation Element / SARS-CoV-2 / COVID-19 / RNA
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsZhang K / Zheludev I
Funding support United States, 7 items
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, 2020-
Header (metadata) releaseAug 19, 2020-
Map releaseAug 19, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xrz
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22296.png

Downloads & links

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Map

FileDownload / File: emd_22296.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.022377597 - 0.07104614
Average (Standard dev.)0.000330831 (±0.0019635276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 137.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z168168168
origin x/y/z0.0000.0000.000
length x/y/z137.760137.760137.760
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS168168168
D min/max/mean-0.0220.0710.000

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Supplemental data

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Sample components

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Entire : 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

EntireName: 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
Components
  • Complex: 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
    • RNA: Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

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Supramolecule #1: 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

SupramoleculeName: 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 28 KDa

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

MacromoleculeName: Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 28.28566 KDa
SequenceString:
GUUUUUAAAC GGGUUUGCGG UGUAAGUGCA GCCCGUCUUA CACCGUGCGG CACAGGCACU AGUACUGAUG UCGUAUACAG GGCUUUUG

GENBANK: GENBANK: MT655131.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 10222 / Average exposure time: 6.0 sec. / Average electron dose: 8.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.2) / Number images used: 109137
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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