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- PDB-8uyj: BtCoV-HKU5 5' proximal stem-loop 5, conformation 4 -

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Entry
Database: PDB / ID: 8uyj
TitleBtCoV-HKU5 5' proximal stem-loop 5, conformation 4
ComponentsBtCoV-HKU5 5' proximal stem-loop 5, conformation 4
KeywordsRNA / SL5 / coronavirus / 5' UTR
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesPipistrellus bat coronavirus HKU5
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.3 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, 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
Other governmentNational Key R&D Program of China 2022YFC2303700
Other governmentNational Key R&D Program of China 2022YFA1302700
Other privateBioX Bowes fellowship
Other privateBioX Interdisciplinary Initiative Program
Other privateChEM-H COVID-19 Drug and Vaccine Prototyping seed grant
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 6, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 13, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

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Assembly

Deposited unit
A: BtCoV-HKU5 5' proximal stem-loop 5, conformation 4


Theoretical massNumber of molelcules
Total (without water)43,5111
Polymers43,5111
Non-polymers00
Water00
1


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

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Components

#1: RNA chain BtCoV-HKU5 5' proximal stem-loop 5, conformation 4


Mass: 43510.676 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pipistrellus bat coronavirus HKU5

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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: BtCoV-HKU5 5' proximal stem-loop 5, conformation 4 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.04373 MDa / Experimental value: NO
Source (natural)Organism: Pipistrellus bat coronavirus HKU5
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium HEPESNa-C8H18N2O4S1
210 mMMagnesium ChlorideMgCl21
SpecimenConc.: 1.31 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 FIELD / Nominal magnification: 96000 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: 4.1 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 18139
Details: Data was collected in two session, 5,728 images were collected with a 4.74s exposure time, 12,411 images where collected with a 3.81s exposure time.
Image scansWidth: 4096 / Height: 4096

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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
12cryoSPARC3.2.0classification3D variability
13cryoSPARC3.2.03D reconstructionNon-uniform Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5109283
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185489 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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