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- EMDB-25550: Map of wt IRES eIF2-containing 48S initiation complex with IRES d... -

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Basic information

Entry
Database: EMDB / ID: EMD-25550
TitleMap of wt IRES eIF2-containing 48S initiation complex with IRES domain II in position 6. Structure 21(wt)
Map data
Sample
  • Complex: 40S ribosomal small subunit with HCV IRES
KeywordsHCV / IRES / 40S / RIBOSOME
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsBrown ZP / Abaeva IS / De S / Hellen CUT / Pestova TV / Frank J
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM55440 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R35GM122602 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI123406 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM139453 United States
CitationJournal: EMBO J / Year: 2022
Title: Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.
Authors: Zuben P Brown / Irina S Abaeva / Swastik De / Christopher U T Hellen / Tatyana V Pestova / Joachim Frank /
Abstract: Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical ...Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.
History
DepositionNov 25, 2021-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25550.png

Downloads & links

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Map

FileDownload / File: emd_25550.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 0.0193
Minimum - Maximum-0.0412852 - 0.096920274
Average (Standard dev.)0.0004979172 (±0.0055323457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 380.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25550_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Refine3D map

Fileemd_25550_additional_1.map
AnnotationRefine3D map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_25550_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_25550_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : 40S ribosomal small subunit with HCV IRES

EntireName: 40S ribosomal small subunit with HCV IRES
Components
  • Complex: 40S ribosomal small subunit with HCV IRES

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Supramolecule #1: 40S ribosomal small subunit with HCV IRES

SupramoleculeName: 40S ribosomal small subunit with HCV IRES / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 2 MDa

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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

Concentration0.000075 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER / Details: H2/O2 mixture for 25 seconds at 25W power
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: 4 second blot time, force 3.

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 4.0 sec. / Average electron dose: 70.9 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Relion 3
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 36155
FSC plot (resolution estimation)

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