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- PDB-8uyp: SARS-CoV-1 5' proximal stem-loop 5 -

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Entry
Database: PDB / ID: 8uyp
TitleSARS-CoV-1 5' proximal stem-loop 5
ComponentsSARS-CoV-1 5' proximal stem-loop 5
KeywordsRNA / SL5 / coronavirus / 5' UTR
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesSARS coronavirus Tor2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsKretsch, R.C. / Xu, L. / Zheludev, I.N. / Zhou, X. / Huang, R. / Nye, G. / Li, S. / Zhang, K. / Chiu, W. / Das, R.
Funding support United States, China, 11items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122579 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129541 United States
National Science Foundation (NSF, United States)GRFPDGE1656518 United States
Ministry of Science and Technology (MoST, China)2022YFC2303700 China
Ministry of Science and Technology (MoST, China)2022YFA1302700 China
Other privateBioX Bowes fellowship United States
Other privateBioX Interdisciplinary Initiative Program United States
Other privateChEM-H COVID-19 Drug and Vaccine Prototyping seed grant United States
Department of Energy (DOE, United States)Coronavirus CARES Act United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)#U19AI171421 United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2024
Title: Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.
Authors: Rachael C Kretsch / Lily Xu / Ivan N Zheludev / Xueting Zhou / Rui Huang / Grace Nye / Shanshan Li / Kaiming Zhang / Wah Chiu / Rhiju Das /
Abstract: Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to ...Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across these human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities and notable differences, with implications for potential protein-binding modes and therapeutic targets.
#1: Journal: bioRxiv / Year: 2023
Title: Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.
Authors: Rachael C Kretsch / Lily Xu / Ivan N Zheludev / Xueting Zhou / Rui Huang / Grace Nye / Shanshan Li / Kaiming Zhang / Wah Chiu / Rhiju Das /
Abstract: Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to ...Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically-determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus SARS-CoV-2, resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4-6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across the studied human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9-8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4-9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities, with implications for potential protein-binding modes and therapeutic targets.
History
DepositionNov 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: SARS-CoV-1 5' proximal stem-loop 5


Theoretical massNumber of molelcules
Total (without water)45,9171
Polymers45,9171
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models8

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Components

#1: RNA chain SARS-CoV-1 5' proximal stem-loop 5


Mass: 45917.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SARS coronavirus Tor2 / References: GenBank: NC_004718.3

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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: SARS-CoV-1 5' proximal stem-loop 5 / Type: COMPLEX / Entity ID: all / Source: SYNTHETIC
Molecular weightValue: 0.04614 MDa / Experimental value: NO
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium HEPESNa-C8H18N2O4S1
210 mMMagnesium ChlorideMgCl21
SpecimenConc.: 1.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.59 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10376
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2.0particle selectionTemplate
2EPUimage acquisition
4cryoSPARC3.2.0CTF correctionPatch CTF Estimation
7Rosetta2020.42model fittingauto-DRRAFTER
9ERRASER2model refinementphenix.real_space_refine
10cryoSPARC3.2.0initial Euler assignmentAb initio reconstruction multi-class
11cryoSPARC3.2.0final Euler assignmentNon-uniform Refinement
12cryoSPARC3.2.0classificationHeterogeneous refinement
13cryoSPARC3.2.03D reconstructionNon-uniform Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1734565
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220917 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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