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- EMDB-42820: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended -

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Entry
Database: EMDB / ID: EMD-42820
TitleSARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
Map dataSARS-CoV-2 RNA SL5-6 domains with SL5b extended.
Sample
  • Complex: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
    • RNA: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
KeywordsSL5 / coronavirus / 5' UTR / RNA
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 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
Ministry of Science and Technology (MoST, China)2022YFC2303700
Ministry of Science and Technology (MoST, 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: 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.
History
DepositionNov 14, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42820.png

Downloads & links

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Map

FileDownload / File: emd_42820.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSARS-CoV-2 RNA SL5-6 domains with SL5b extended.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 512 pix.
= 379.392 Å		0.74 Å/pix.
x 512 pix.
= 379.392 Å		0.74 Å/pix.
x 512 pix.
= 379.392 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.741 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.32
Minimum - Maximum-0.3342343 - 0.98034716
Average (Standard dev.)0.00024540388 (±0.014409452)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 379.392 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42820_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 the SARS-CoV-2 RNA SL5-6 domains with SL5b extended.

Fileemd_42820_half_map_1.map
AnnotationHalf map of the SARS-CoV-2 RNA SL5-6 domains with SL5b extended.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the SARS-CoV-2 RNA SL5-6 domains with SL5b extended.

Fileemd_42820_half_map_2.map
AnnotationHalf map of the SARS-CoV-2 RNA SL5-6 domains with SL5b extended.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended

EntireName: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
Components
  • Complex: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
    • RNA: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended

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Supramolecule #1: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended

SupramoleculeName: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 65.7 KDa

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Macromolecule #1: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended

MacromoleculeName: SARS-CoV-2 5' proximal stem-loop 5 and 6 with SL5b extended
type: rna / ID: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
SequenceString: UGUCGUUGAC AGGACACGAG UAACUCGUCU AUCUUCUGCA GGCUGCUUAC GGUUUCGUCC GUGUUGCAGC CGAUCAUCAG CACAUCUAGC UGGGUUUCGU CCCAGCGGGU GUGACCGAAA GGUAAGAUGG AGAGCCUUGU CCCUGGUUUC AACGAGAAAA CACACGUCCA ...String:
UGUCGUUGAC AGGACACGAG UAACUCGUCU AUCUUCUGCA GGCUGCUUAC GGUUUCGUCC GUGUUGCAGC CGAUCAUCAG CACAUCUAGC UGGGUUUCGU CCCAGCGGGU GUGACCGAAA GGUAAGAUGG AGAGCCUUGU CCCUGGUUUC AACGAGAAAA CACACGUCCA ACUCAGUUUG CCUGUUUUAC AGGUUCGCGA CGUG

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

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
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 11496 / Average exposure time: 4.66 sec. / Average electron dose: 50.0 e/Å2
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: 165000
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: 810035
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Non-uniform Refinement / Number images used: 199745
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: 3 / Avg.num./class: 178343 / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Heterogeneous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER

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