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- PDB-7ado: Cryo-EM structure of human ER membrane protein complex in lipid n... -

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Basic information

Entry
Database: PDB / ID: 7ado
TitleCryo-EM structure of human ER membrane protein complex in lipid nanodiscs
Components
  • (ER membrane protein complex subunit ...Endoplasmic reticulum) x 8
  • Membrane magnesium transporter 1
  • Unassigned helix
KeywordsMEMBRANE PROTEIN / ER membrane protein / EMC / Membrane protein biogenesis
Function / homology
Function and homology information


inorganic cation transmembrane transporter activity / extrinsic component of endoplasmic reticulum membrane / EMC complex / omegasome membrane / protein insertion into ER membrane by stop-transfer membrane-anchor sequence / magnesium ion transport / cobalt ion transmembrane transporter activity / tail-anchored membrane protein insertion into ER membrane / Miscellaneous transport and binding events / magnesium ion transmembrane transporter activity ...inorganic cation transmembrane transporter activity / extrinsic component of endoplasmic reticulum membrane / EMC complex / omegasome membrane / protein insertion into ER membrane by stop-transfer membrane-anchor sequence / magnesium ion transport / cobalt ion transmembrane transporter activity / tail-anchored membrane protein insertion into ER membrane / Miscellaneous transport and binding events / magnesium ion transmembrane transporter activity / ferrous iron transmembrane transporter activity / copper ion transport / autophagosome assembly / RHOA GTPase cycle / positive regulation of endothelial cell proliferation / positive regulation of endothelial cell migration / positive regulation of angiogenesis / early endosome membrane / carbohydrate binding / angiogenesis / early endosome / Golgi membrane / apoptotic process / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / protein-containing complex / extracellular region / membrane / plasma membrane / cytoplasm
Similarity search - Function
ER membrane protein complex subunit 8/9 / Uncharacterised protein family (UPF0172) / TMEM85/ER membrane protein complex subunit 4 / Protein of unknown function (DUF1077) / ER membrane protein complex subunit 7, beta-sandwich domain / ER membrane protein complex subunit 7 / ER membrane protein complex subunit 7, beta-sandwich domain / ER membrane protein complex subunit 6 / ER membrane protein complex subunit 3 / ER membrane protein complex subunit 1, C-terminal ...ER membrane protein complex subunit 8/9 / Uncharacterised protein family (UPF0172) / TMEM85/ER membrane protein complex subunit 4 / Protein of unknown function (DUF1077) / ER membrane protein complex subunit 7, beta-sandwich domain / ER membrane protein complex subunit 7 / ER membrane protein complex subunit 7, beta-sandwich domain / ER membrane protein complex subunit 6 / ER membrane protein complex subunit 3 / ER membrane protein complex subunit 1, C-terminal / Membrane magnesium transporter / ER membrane protein complex subunit 1 / ER membrane protein complex subunit 6-like / EMC6 / ER membrane protein complex subunit 1, C-terminal / Membrane magnesium transporter / ER membrane protein complex subunit 10 / ER membrane protein complex subunit 2-like / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein DUF106 / Pyrrolo-quinoline quinone repeat / PQQ-like domain / Carbohydrate-binding-like fold / Tetratricopeptide repeat / Quinoprotein alcohol dehydrogenase-like superfamily / TPR repeat region circular profile. / TPR repeat profile. / MPN domain / MPN domain profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / ER membrane protein complex subunit 8 / ER membrane protein complex subunit 2 / ER membrane protein complex subunit 4 / ER membrane protein complex subunit 10 / ER membrane protein complex subunit 5 / ER membrane protein complex subunit 1 / ER membrane protein complex subunit 6 / Endoplasmic reticulum membrane protein complex subunit 7 / ER membrane protein complex subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsBraeuning, B. / Prabu, J.R. / Miller-Vedam, L.E. / Weissman, J.S. / Frost, A. / Schulman, B.A.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)SCHU 3196/1-1 Germany
Max Planck Society Germany
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
DepositionSep 15, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 2.0Jan 20, 2021Group: Advisory / Atomic model / Structure summary
Category: atom_site / pdbx_poly_seq_scheme ...atom_site / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_polymer_linkage
Item: _atom_site.auth_comp_id / _atom_site.auth_seq_id ..._atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_comp_id / _atom_site.label_seq_id / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_unobs_or_zero_occ_atoms.auth_atom_id / _pdbx_unobs_or_zero_occ_atoms.auth_comp_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_atom_id / _pdbx_unobs_or_zero_occ_atoms.label_comp_id / _pdbx_unobs_or_zero_occ_atoms.label_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_comp_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.label_comp_id / _pdbx_unobs_or_zero_occ_residues.label_seq_id

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Structure visualization

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Assembly

Deposited unit
A: ER membrane protein complex subunit 1
B: ER membrane protein complex subunit 2
C: ER membrane protein complex subunit 3
D: ER membrane protein complex subunit 4
E: Membrane magnesium transporter 1
F: ER membrane protein complex subunit 6
G: ER membrane protein complex subunit 7
H: ER membrane protein complex subunit 8
I: ER membrane protein complex subunit 10
K: Unassigned helix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)308,54419
Polymers303,72310
Non-polymers4,8209
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37550 Å2
ΔGint-222 kcal/mol
Surface area95580 Å2
MethodPISA

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Components

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ER membrane protein complex subunit ... , 8 types, 8 molecules ABCDFGHI

#1: Protein ER membrane protein complex subunit 1 / Endoplasmic reticulum


Mass: 111886.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC1, KIAA0090, PSEC0263 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q8N766
#2: Protein ER membrane protein complex subunit 2 / Endoplasmic reticulum / Tetratricopeptide repeat protein 35 / TPR repeat protein 35


Mass: 34882.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC2, KIAA0103, TTC35 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q15006
#3: Protein ER membrane protein complex subunit 3 / Endoplasmic reticulum / Transmembrane protein 111


Mass: 29981.924 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC3, TMEM111 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9P0I2
#4: Protein ER membrane protein complex subunit 4 / Endoplasmic reticulum / Cell proliferation-inducing gene 17 protein / Transmembrane protein 85


Mass: 20104.572 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: There is no mutations but sequence from model and input are not aligning well.
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC4, TMEM85, HSPC184, PIG17 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q5J8M3
#6: Protein ER membrane protein complex subunit 6 / Endoplasmic reticulum / Transmembrane protein 93


Mass: 12029.248 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC6, TMEM93 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9BV81
#7: Protein ER membrane protein complex subunit 7 / Endoplasmic reticulum


Mass: 26501.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC7, C11orf3, C15orf24, HT022, UNQ905/PRO1926 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9NPA0
#8: Protein ER membrane protein complex subunit 8 / Endoplasmic reticulum / Neighbor of COX4 / Protein FAM158B


Mass: 23807.076 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Map modelled as EMC8; both EMC8 and its paralog EMC9 (44% identity) were co-expressed and turns out they can both occupy same position on complex in mutually exclusive manner. We chose to ...Details: Map modelled as EMC8; both EMC8 and its paralog EMC9 (44% identity) were co-expressed and turns out they can both occupy same position on complex in mutually exclusive manner. We chose to model as EMC8 but note that map is likely superposition of both.
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC8, C16orf2, C16orf4, COX4AL, COX4NB, FAM158B, NOC4 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: O43402
#9: Protein ER membrane protein complex subunit 10 / Endoplasmic reticulum / Hematopoietic signal peptide-containing membrane domain-containing protein 1


Mass: 27446.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC10, C19orf63, HSM1, INM02, UNQ764/PRO1556 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q5UCC4

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Protein / Protein/peptide / Sugars / Non-polymers , 4 types, 11 molecules EK

#10: Protein/peptide Unassigned helix


Mass: 1379.692 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This is transmembrane alpha-helix which was modeled as poly-alanine.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pEG / Production host: Homo sapiens (human)
#11: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#12: Chemical
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#5: Protein Membrane magnesium transporter 1 / ER membrane protein complex subunit 5 / Transmembrane protein 32


Mass: 15703.762 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MMGT1, EMC5, TMEM32 / Plasmid: pEG / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q8N4V1

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human endoplasmic reticulum membrane protein complex (EMC) in POPC nanodiscs
Type: COMPLEX / Entity ID: #1-#10 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 GnTI- / Plasmid: pEG
Buffer solutionpH: 6
Details: 10 mM ammonium citrate pH 6.0, 100 mM sodium chloride, 0.25 mM TCEP
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Dec 23, 2019
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 3 sec. / Electron dose: 72 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
7Cootmodel fitting
12RELION3D reconstruction
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177560 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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