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- EMDB-43137: SARS-CoV-2 Frameshift Stimulatory Element with Upstream Multibran... -

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Basic information

Entry
Database: EMDB / ID: EMD-43137
TitleSARS-CoV-2 Frameshift Stimulatory Element with Upstream Multibranch Loop
Map data
Sample
  • Virus: Severe acute respiratory syndrome coronavirus
    • RNA: Frameshift Stimulatory Element with Upstream Multi-branch Loop
KeywordsRNA / Coronavirus / SARS-CoV-2 / Programmed -1 Ribosomal Frameshifting / -1 PRF / Frameshift Stimulatory Element / FSE / Attenuator Hairpin
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Severe acute respiratory syndrome coronavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsPeterson JM / Becker ST / O'Leary CA / Juneja P / Yang Y / Moss WN
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM133810 United States
CitationJournal: Biochemistry / Year: 2024
Title: Structure of the SARS-CoV-2 Frameshift Stimulatory Element with an Upstream Multibranch Loop.
Authors: Jake M Peterson / Scott T Becker / Collin A O'Leary / Puneet Juneja / Yang Yang / Walter N Moss /
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The ...The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to -1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site-a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.
History
DepositionDec 14, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43137.png

Downloads & links

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Map

FileDownload / File: emd_43137.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.133 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.11588581 - 0.1850674
Average (Standard dev.)0.00008037884 (±0.0065108426)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 226.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43137_msk_1.map
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AxesZYX

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Additional map: #1

Fileemd_43137_additional_1.map
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Half map: #1

Fileemd_43137_half_map_1.map
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Half map: #2

Fileemd_43137_half_map_2.map
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Sample components

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Entire : Severe acute respiratory syndrome coronavirus

EntireName: Severe acute respiratory syndrome coronavirus
Components
  • Virus: Severe acute respiratory syndrome coronavirus
    • RNA: Frameshift Stimulatory Element with Upstream Multi-branch Loop

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Supramolecule #1: Severe acute respiratory syndrome coronavirus

SupramoleculeName: Severe acute respiratory syndrome coronavirus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / Details: PCR amplified and transcribed using T7 polymerase. / NCBI-ID: 2901879
Sci species name: Severe acute respiratory syndrome coronavirus
Sci species strain: Wuhan-Hu-1 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human) / Strain: Wuhan-Hu-1

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Macromolecule #1: Frameshift Stimulatory Element with Upstream Multi-branch Loop

MacromoleculeName: Frameshift Stimulatory Element with Upstream Multi-branch Loop
type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 37.90141 KDa
SequenceString:
GGGAACCCAU GCUUCAGUCA GCUGAUGCAC AAUCGUUUUU AAACGGGUUU CCGGUGUAAG UGCAGCCCGU CUUACACCGU GCGGCACAG GCACUAGUAC UGAUGUCGUA UACAGGGCU

GENBANK: GENBANK: NC_045512.2

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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

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 3 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 36000

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 627438
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 600000 / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8vci:
SARS-CoV-2 Frameshift Stimulatory Element with Upstream Multibranch Loop

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