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- EMDB-19199: Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin... -

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Basic information

Entry
Database: EMDB / ID: EMD-19199
TitleStructure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, bound TRAP-alpha TMD
Map data
Sample
  • Complex: 80S ribosome translating a stalled, two-TMD nascent chain (derived from rhodopsin), and bound to Sec61, the TRAP complex, and RAMP4
KeywordsRibosome / Membrane protein / Translocon / Transport
Biological speciesCanis lupus familiaris (dog)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69243 Å
AuthorsLewis AJO / Hegde RS
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A022_1007 United Kingdom
CitationJournal: Elife / Year: 2024
Title: Structural analysis of the dynamic ribosome-translocon complex.
Authors: Aaron J O Lewis / Frank Zhong / Robert J Keenan / Ramanujan S Hegde /
Abstract: The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane ...The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.
History
DepositionDec 20, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19199.png

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Map

FileDownload / File: emd_19199.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 420 pix.
= 562.716 Å		1.34 Å/pix.
x 420 pix.
= 562.716 Å		1.34 Å/pix.
x 420 pix.
= 562.716 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3398 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0231
Minimum - Maximum-0.07814522 - 0.24408749
Average (Standard dev.)0.0005007235 (±0.0067392085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 562.716 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_19199_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_19199_half_map_2.map
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Sample components

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Entire : 80S ribosome translating a stalled, two-TMD nascent chain (derive...

EntireName: 80S ribosome translating a stalled, two-TMD nascent chain (derived from rhodopsin), and bound to Sec61, the TRAP complex, and RAMP4
Components
  • Complex: 80S ribosome translating a stalled, two-TMD nascent chain (derived from rhodopsin), and bound to Sec61, the TRAP complex, and RAMP4

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Supramolecule #1: 80S ribosome translating a stalled, two-TMD nascent chain (derive...

SupramoleculeName: 80S ribosome translating a stalled, two-TMD nascent chain (derived from rhodopsin), and bound to Sec61, the TRAP complex, and RAMP4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#56 / Details: Sample prepared by in vitro translation
Source (natural)Organism: Canis lupus familiaris (dog)

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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

BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMHEPES-KOH buffer, pH 7.5
200.0 mMpotassium acetateKOAc
5.0 mMmagnesium acetateMg(OAc)2
0.25 % (w/w)digitonin
50.0 mMbiotin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 17540 / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.9000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.69243 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 121515
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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