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- EMDB-42802: BtCoV-HKU5 5' proximal stem-loop 5, conformation 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-42802
TitleBtCoV-HKU5 5' proximal stem-loop 5, conformation 3
Map dataConformation 3 of the BtCoV-HKU5 RNA SL5 domain.
Sample
  • Complex: BtCoV-HKU5 5' proximal stem-loop 5, conformation 3
    • RNA: BtCoV-HKU5 5' proximal stem-loop 5
KeywordsSL5 / coronavirus / 5' UTR / RNA
Biological speciesPipistrellus bat coronavirus HKU5
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsKretsch RC / Xu L / Zheludev IN / Zhou X / Huang R / Nye G / Li S / Zhang K / Chiu W / Das R
Funding support United States, 11 items
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
CitationJournal: 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, 2023-
Header (metadata) releaseDec 6, 2023-
Map releaseDec 6, 2023-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42802.png

Downloads & links

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Map

FileDownload / File: emd_42802.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConformation 3 of the BtCoV-HKU5 RNA SL5 domain.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 200 pix.
= 164. Å		0.82 Å/pix.
x 200 pix.
= 164. Å		0.82 Å/pix.
x 200 pix.
= 164. 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: 0.164
Minimum - Maximum-0.23320073 - 1.21045
Average (Standard dev.)0.0034966185 (±0.03611458)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 164.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42802_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of conformation 3 of the BtCoV-HKU5 RNA SL5 domain.

Fileemd_42802_half_map_1.map
AnnotationHalf map of conformation 3 of the BtCoV-HKU5 RNA SL5 domain.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of conformation 3 of the BtCoV-HKU5 RNA SL5 domain.

Fileemd_42802_half_map_2.map
AnnotationHalf map of conformation 3 of the BtCoV-HKU5 RNA SL5 domain.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BtCoV-HKU5 5' proximal stem-loop 5, conformation 3

EntireName: BtCoV-HKU5 5' proximal stem-loop 5, conformation 3
Components
  • Complex: BtCoV-HKU5 5' proximal stem-loop 5, conformation 3
    • RNA: BtCoV-HKU5 5' proximal stem-loop 5

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Supramolecule #1: BtCoV-HKU5 5' proximal stem-loop 5, conformation 3

SupramoleculeName: BtCoV-HKU5 5' proximal stem-loop 5, conformation 3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pipistrellus bat coronavirus HKU5
Molecular weightTheoretical: 43.73 KDa

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Macromolecule #1: BtCoV-HKU5 5' proximal stem-loop 5

MacromoleculeName: BtCoV-HKU5 5' proximal stem-loop 5 / type: rna / ID: 1
Source (natural)Organism: Pipistrellus bat coronavirus HKU5
SequenceString:
GGAGCAUCGU GUCUCAAGUG CUUCACGGUC ACAAUAUACC GUUUCGUCGG GUGCGUGGCA AUUCGGUGCA CAUCAUGUCU UUCGUGGCUG GUGUGGCUCC UCAAGGUGCG AGGGGCAAGU AUAGAGCAGA GCUCC

GENBANK: GENBANK: NC_009020.1

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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

Concentration1.31 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMNa-C8H18N2O4SSodium HEPES
10.0 mMMgCl2Magnesium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 18139 / Average exposure time: 4.1 sec. / Average electron dose: 50.0 e/Å2
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.
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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 96000
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

Particle selectionNumber selected: 5109283
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Non-uniform Refinement / Number images used: 127548
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Ab initio reconstruction multi-class
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Non-uniform Refinement
Final 3D classificationNumber classes: 4 / Avg.num./class: 166600 / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: 3D variability
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER

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