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- PDB-7adp: Cryo-EM structure of human ER membrane protein complex in GDN det... -

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Basic information

Entry
Database: PDB / ID: 7adp
TitleCryo-EM structure of human ER membrane protein complex in GDN detergent
Components
  • (ER membrane protein complex subunit ...) x 7
  • Membrane magnesium transporter 1
KeywordsMEMBRANE PROTEIN / ER membrane protein / EMC / Membrane protein biogenesis
Function / homology
Function and homology information


extrinsic component of endoplasmic reticulum membrane / inorganic cation transmembrane transporter activity / EMC complex / omegasome membrane / protein insertion into ER membrane by stop-transfer membrane-anchor sequence / magnesium ion transport / cobalt ion transmembrane transporter activity / Miscellaneous transport and binding events / tail-anchored membrane protein insertion into ER membrane / magnesium ion transmembrane transporter activity ...extrinsic component of endoplasmic reticulum membrane / inorganic cation transmembrane transporter activity / EMC complex / omegasome membrane / protein insertion into ER membrane by stop-transfer membrane-anchor sequence / magnesium ion transport / cobalt ion transmembrane transporter activity / Miscellaneous transport and binding events / tail-anchored membrane protein insertion into ER membrane / magnesium ion transmembrane transporter activity / ferrous iron transmembrane transporter activity / protein folding in endoplasmic reticulum / 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 / 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 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 ...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 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 / 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
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 / ER membrane protein complex subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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

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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
H: ER membrane protein complex subunit 8
I: ER membrane protein complex subunit 10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,50611
Polymers275,8428
Non-polymers6643
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28260 Å2
ΔGint-169 kcal/mol
Surface area88430 Å2
MethodPISA

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Components

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ER membrane protein complex subunit ... , 7 types, 7 molecules ABCDFHI

#1: Protein ER membrane protein complex subunit 1


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): HEK 293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q8N766
#2: Protein ER membrane protein complex subunit 2 / 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): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q15006
#3: Protein ER membrane protein complex subunit 3 / 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): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9P0I2
#4: Protein ER membrane protein complex subunit 4 / 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: No mutation; again, as for D_1292111207 there is failed alignment between structure and sequence. Please see communication, thanks!
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC4, TMEM85, HSPC184, PIG17 / Plasmid: pEG / Cell line (production host): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q5J8M3
#6: Protein ER membrane protein complex subunit 6 / 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): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9BV81
#7: Protein ER membrane protein complex subunit 8 / Neighbor of COX4 / Protein FAM158B


Mass: 23807.076 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EMC8, C16orf2, C16orf4, COX4AL, COX4NB, FAM158B, NOC4 / Plasmid: pEG / Cell line (production host): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: O43402
#8: Protein ER membrane protein complex subunit 10 / 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): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q5UCC4

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Protein / Sugars , 2 types, 4 molecules E

#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): HEK 293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q8N4V1
#9: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / 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

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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-#8 / 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
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: Oct 16, 2019
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 5 sec. / Electron dose: 62 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameCategory
7Cootmodel fitting
9PHENIXmodel refinement
13RELION3D reconstruction
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144222 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00530340
ELECTRON MICROSCOPYf_angle_d0.71854779
ELECTRON MICROSCOPYf_dihedral_angle_d10.8312199
ELECTRON MICROSCOPYf_chiral_restr0.0462362
ELECTRON MICROSCOPYf_plane_restr0.0044409

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