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- EMDB-21426: Single Particle Cryo-EM Structure of the Natively Isolated Sec61 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21426
TitleSingle Particle Cryo-EM Structure of the Natively Isolated Sec61 complex, TMCO1, Nicalin, TMEM147, and CCDC47 Containing Translocon
Map data
Sample
  • Complex: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1
    • Complex: Translocon
      • Complex: Sec61 Complex
        • Complex: Sec61 alpha
        • Complex: Sec61 beta
        • Complex: Sec61 gamma
      • Complex: TMCO1
      • Complex: NOMO-Nicalin-TMEM147 Complex
        • Complex: TMEM147
        • Complex: Nicalin
        • Complex: NOMO
      • Complex: CCDC47
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsMcGilvray PT / Anghel SA / Sundaram A / Trnka MJ / Zhong F / Hu H / Burlingame AL / Keenan RJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM130051-01 United States
CitationJournal: Elife / Year: 2020
Title: An ER translocon for multi-pass membrane protein biogenesis.
Authors: Philip T McGilvray / S Andrei Anghel / Arunkumar Sundaram / Frank Zhong / Michael J Trnka / James R Fuller / Hong Hu / Alma L Burlingame / Robert J Keenan /
Abstract: Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here ...Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.
History
DepositionFeb 21, 2020-
Header (metadata) releaseMar 25, 2020-
Map releaseSep 2, 2020-
UpdateSep 2, 2020-
Current statusSep 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0095
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0095
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21426.png

Downloads & links

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Map

FileDownload / File: emd_21426.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.0095 / Movie #1: 0.0095
Minimum - Maximum-0.021484276 - 0.05110217
Average (Standard dev.)-0.00002623170 (±0.0009071877)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00500
Dimensions500500500
Spacing500500500
CellA=B=C: 680.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z500500500
origin x/y/z0.0000.0000.000
length x/y/z680.000680.000680.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00500
NC/NR/NS500500500
D min/max/mean-0.0210.051-0.000

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Supplemental data

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Mask #1

Fileemd_21426_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_21426_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_21426_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Native complex of a translating ribosome and a novel translocon c...

EntireName: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1
Components
  • Complex: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1
    • Complex: Translocon
      • Complex: Sec61 Complex
        • Complex: Sec61 alpha
        • Complex: Sec61 beta
        • Complex: Sec61 gamma
      • Complex: TMCO1
      • Complex: NOMO-Nicalin-TMEM147 Complex
        • Complex: TMEM147
        • Complex: Nicalin
        • Complex: NOMO
      • Complex: CCDC47

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Supramolecule #1: Native complex of a translating ribosome and a novel translocon c...

SupramoleculeName: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum
Molecular weightTheoretical: 140 KDa

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Supramolecule #2: Translocon

SupramoleculeName: Translocon / type: complex / ID: 2 / Parent: 1

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Supramolecule #3: Sec61 Complex

SupramoleculeName: Sec61 Complex / type: complex / ID: 3 / Parent: 2 / Details: Sec61alpha, beta, and gamma
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #4: TMCO1

SupramoleculeName: TMCO1 / type: complex / ID: 4 / Parent: 2
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #5: NOMO-Nicalin-TMEM147 Complex

SupramoleculeName: NOMO-Nicalin-TMEM147 Complex / type: complex / ID: 5 / Parent: 2
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #6: CCDC47

SupramoleculeName: CCDC47 / type: complex / ID: 6 / Parent: 2
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #7: Sec61 alpha

SupramoleculeName: Sec61 alpha / type: complex / ID: 7 / Parent: 3
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #8: Sec61 beta

SupramoleculeName: Sec61 beta / type: complex / ID: 8 / Parent: 3
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #9: Sec61 gamma

SupramoleculeName: Sec61 gamma / type: complex / ID: 9 / Parent: 3
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #10: TMEM147

SupramoleculeName: TMEM147 / type: complex / ID: 10 / Parent: 5
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #11: Nicalin

SupramoleculeName: Nicalin / type: complex / ID: 11 / Parent: 5
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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Supramolecule #12: NOMO

SupramoleculeName: NOMO / type: complex / ID: 12 / Parent: 5
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

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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

Concentration0.4 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMC2H3KO2potassium acetate
50.0 mMC8H18N2O4SHEPES, pH 7.4
5.0 mMMgCl2magnesium chloride
0.25 percent (w/v)C56H92O29digitonin
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: Two filter papers were added to each arm, 2.5 microliters of sample were added to grids, samples were blotted for 11 seconds, and 0.5 second of drain time was allowed before vitrification..
DetailsSample was well-dispersed on a thin (~2 nm) carbon film.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 5562 / Average exposure time: 3.8 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1049128
CTF correctionSoftware - Name: Gctf (ver. 0.5)
Startup modelType of model: EMDB MAP
EMDB ID:

5592


Details: EMD-5592, 60S ribosome low pass-filtered to 50 Angstrom resolution
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 9 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 82684
FSC plot (resolution estimation)

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