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- EMDB-23033: Cryo-EM structure of Saccharomyces cerevisiae ER membrane protein... -

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Basic information

Entry
Database: EMDB / ID: EMD-23033
TitleCryo-EM structure of Saccharomyces cerevisiae ER membrane protein complex bound to a Fab in DDM detergent
Map data
SampleS. cerevisiae endoplasmic reticulum membrane protein complex (EMC) bound to Fab in DDM detergent
  • endoplasmic reticulum membrane protein complex
  • Fab DH4
  • (ER membrane protein complex subunit ...Endoplasmic reticulum) x 6
  • Protein SOP4
  • Endoplasmic reticulum membrane protein complex subunit 10
  • Fab DH4 heavy chain
  • Fab DH4 light chain
  • Unassigned helix
  • ligand
Function / homology
Function and homology information


protein insertion into ER membrane by stop-transfer membrane-anchor sequence / EMC complex / protein folding in endoplasmic reticulum / autophagosome assembly / endoplasmic reticulum to Golgi vesicle-mediated transport / protein transport / chaperone binding / endoplasmic reticulum membrane / endoplasmic reticulum / integral component of membrane / nucleus
Similarity search - Function
Protein Sop4 / Suppressor of PMA 1-7 protein / Protein of unknown function (DUF1077) / TMEM85/ER membrane protein complex subunit 4 / Membrane magnesium transporter / ER membrane protein complex subunit 1, C-terminal / ER membrane protein complex subunit 3 / ER membrane protein complex subunit 6-like / EMC6 / ER membrane protein complex subunit 1, C-terminal ...Protein Sop4 / Suppressor of PMA 1-7 protein / Protein of unknown function (DUF1077) / TMEM85/ER membrane protein complex subunit 4 / Membrane magnesium transporter / ER membrane protein complex subunit 1, C-terminal / ER membrane protein complex subunit 3 / ER membrane protein complex subunit 6-like / EMC6 / ER membrane protein complex subunit 1, C-terminal / Membrane magnesium transporter / ER membrane protein complex subunit 1 / ER membrane protein complex subunit 2-like / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein DUF106 / TPR repeat profile. / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
ER membrane protein complex subunit 1 / ER membrane protein complex subunit 3 / Protein SOP4 / ER membrane protein complex subunit 5 / ER membrane protein complex subunit 2 / ER membrane protein complex subunit 4 / Endoplasmic reticulum membrane protein complex subunit 10 / ER membrane protein complex subunit 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4743 (yeast) / Homo sapiens (human) / Baker's yeast (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsMiller-Vedam LE / Schirle Oakdale NS / Braeuning B / Boydston EA / Sevillano N / Popova KD / Bonnar JL / Shurtleff MJ / Prabu JR / Stroud RM ...Miller-Vedam LE / Schirle Oakdale NS / Braeuning B / Boydston EA / Sevillano N / Popova KD / Bonnar JL / Shurtleff MJ / Prabu JR / Stroud RM / Craik CS / Schulman BA / Weissman JS / Frost A
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/Office of the Director1DP2OD017690-01 United States
CitationJournal: Elife / Year: 2020
Title: Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients.
Authors: Lakshmi E Miller-Vedam / Bastian Bräuning / Katerina D Popova / Nicole T Schirle Oakdale / Jessica L Bonnar / Jesuraj R Prabu / Elizabeth A Boydston / Natalia Sevillano / Matthew J ...Authors: Lakshmi E Miller-Vedam / Bastian Bräuning / Katerina D Popova / Nicole T Schirle Oakdale / Jessica L Bonnar / Jesuraj R Prabu / Elizabeth A Boydston / Natalia Sevillano / Matthew J Shurtleff / Robert M Stroud / Charles S Craik / Brenda A Schulman / Adam Frost / Jonathan S Weissman /
Abstract: Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player ...Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.
History
DepositionNov 24, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7ktx
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23033.map.gz / Format: CCP4 / Size: 71.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 266 pix.
= 359.1 Å
1.35 Å/pix.
x 266 pix.
= 359.1 Å
1.35 Å/pix.
x 266 pix.
= 359.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.06927432 - 0.1280162
Average (Standard dev.)0.0002912958 (±0.003309046)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions266266266
Spacing266266266
CellA=B=C: 359.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z266266266
origin x/y/z0.0000.0000.000
length x/y/z359.100359.100359.100
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS266266266
D min/max/mean-0.0690.1280.000

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Supplemental data

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Segmentation: #1

Fileemd_23033_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: unsharpened map

Fileemd_23033_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map 2

Fileemd_23033_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map 1

Fileemd_23033_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire S. cerevisiae endoplasmic reticulum membrane protein complex (EMC...

EntireName: S. cerevisiae endoplasmic reticulum membrane protein complex (EMC) bound to Fab in DDM detergent
Number of Components: 15

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Component #1: protein, S. cerevisiae endoplasmic reticulum membrane protein com...

ProteinName: S. cerevisiae endoplasmic reticulum membrane protein complex (EMC) bound to Fab in DDM detergent
Recombinant expression: No

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Component #2: protein, endoplasmic reticulum membrane protein complex

ProteinName: endoplasmic reticulum membrane protein complex / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4741

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Component #3: protein, Fab DH4

ProteinName: Fab DH4 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: BL21-Gold(DE3)pLysS AG

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Component #4: protein, ER membrane protein complex subunit 1

ProteinName: ER membrane protein complex subunit 1Endoplasmic reticulum
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 87.272938 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #5: protein, ER membrane protein complex subunit 2

ProteinName: ER membrane protein complex subunit 2Endoplasmic reticulum
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 33.893211 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #6: protein, ER membrane protein complex subunit 3

ProteinName: ER membrane protein complex subunit 3Endoplasmic reticulum
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.372842 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #7: protein, ER membrane protein complex subunit 4

ProteinName: ER membrane protein complex subunit 4Endoplasmic reticulum
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 21.478721 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #8: protein, ER membrane protein complex subunit 5

ProteinName: ER membrane protein complex subunit 5Endoplasmic reticulum
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 20.489887 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #9: protein, ER membrane protein complex subunit 6

ProteinName: ER membrane protein complex subunit 6Endoplasmic reticulum
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 12.411359 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #10: protein, Protein SOP4

ProteinName: Protein SOP4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.627627 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #11: protein, Endoplasmic reticulum membrane protein complex subunit 10

ProteinName: Endoplasmic reticulum membrane protein complex subunit 10
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 22.792824 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #12: protein, Fab DH4 heavy chain

ProteinName: Fab DH4 heavy chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.849994 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #13: protein, Fab DH4 light chain

ProteinName: Fab DH4 light chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.566648 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #14: protein, Unassigned helix

ProteinName: Unassigned helix / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.060531 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BY4743

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Component #15: ligand, 2-acetamido-2-deoxy-beta-D-glucopyranose

LigandName: 2-acetamido-2-deoxy-beta-D-glucopyranose / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionpH: 6.8
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen Name: ETHANE / Humidity: 100 %

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Electron microscopy imaging #1

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Details: Dataset 1 collected on FEI Polara at UCSF on August 22nd, 2016.
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 56.8 e/Å2 / Illumination Mode: FLOOD BEAM
LensImaging Mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Electron microscopy imaging #2

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: Dataset 2 collected on FEI Titan Krios at Janelia Research Campus on October 20th, 2016.
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 58.3 e/Å2 / Illumination Mode: FLOOD BEAM
LensImaging Mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of Projections: 83599
3D reconstruction #1Resolution: 4.3 Å / Resolution Method: FSC 0.143 CUT-OFF
Details: These two datasets were processed independently initially. Upon selecting the best subset of data and then re-extracted and scaled to the same pixel size and combined to get to the final 4.3 ...Details: These two datasets were processed independently initially. Upon selecting the best subset of data and then re-extracted and scaled to the same pixel size and combined to get to the final 4.3 A resolution maps and model.
3D reconstruction #2Resolution: 4.3 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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