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- EMDB-22297: Nanostructure of Frameshift Stimulation Element Tagged by ATP-TTR3 -

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Entry
Database: EMDB / ID: EMD-22297
TitleNanostructure of Frameshift Stimulation Element Tagged by ATP-TTR3
Map dataNanostructure of Frameshift Stimulation Element tagged by ATP-TTR3
Sample
  • Complex: Frameshift Stimulation Element tagged by ATP-TTR3
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 6.4 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, 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)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
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-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22297.png

Downloads & links

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Map

FileDownload / File: emd_22297.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNanostructure of Frameshift Stimulation Element tagged by ATP-TTR3
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.51709306 - 0.89222866
Average (Standard dev.)0.0021696864 (±0.025876217)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z246.000246.000246.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.5170.8920.002

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Supplemental data

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Sample components

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Entire : Frameshift Stimulation Element tagged by ATP-TTR3

EntireName: Frameshift Stimulation Element tagged by ATP-TTR3
Components
  • Complex: Frameshift Stimulation Element tagged by ATP-TTR3

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Supramolecule #1: Frameshift Stimulation Element tagged by ATP-TTR3

SupramoleculeName: 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 weightTheoretical: 60 KDa

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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
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.13)
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Number images used: 257558

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