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- PDB-7ktx: Cryo-EM structure of Saccharomyces cerevisiae ER membrane protein... -

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Basic information

Entry
Database: PDB / ID: 7ktx
TitleCryo-EM structure of Saccharomyces cerevisiae ER membrane protein complex bound to a Fab in DDM detergent
Components
  • (ER membrane protein complex subunit ...) x 6
  • Endoplasmic reticulum membrane protein complex subunit 10
  • Fab DH4 heavy chain
  • Fab DH4 light chain
  • Protein SOP4
  • Unassigned helix
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / ER membrane protein complex / EMC / membrane protein biogenesis / insertase / chaperone / endoplasmic / reticulum / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


EMC complex / protein insertion into ER membrane by stop-transfer membrane-anchor sequence / protein folding in endoplasmic reticulum / phospholipid transport / autophagosome assembly / endoplasmic reticulum to Golgi vesicle-mediated transport / phospholipid metabolic process / protein transport / protein-folding chaperone binding / endoplasmic reticulum membrane ...EMC complex / protein insertion into ER membrane by stop-transfer membrane-anchor sequence / protein folding in endoplasmic reticulum / phospholipid transport / autophagosome assembly / endoplasmic reticulum to Golgi vesicle-mediated transport / phospholipid metabolic process / protein transport / protein-folding chaperone binding / endoplasmic reticulum membrane / endoplasmic reticulum / nucleus / membrane
Similarity search - Function
Protein Sop4 / : / Suppressor of PMA 1-7 protein / TMEM85/ER membrane protein complex subunit 4 / ER membrane protein complex subunit 4 / 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 ...Protein Sop4 / : / Suppressor of PMA 1-7 protein / TMEM85/ER membrane protein complex subunit 4 / ER membrane protein complex subunit 4 / 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 / 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 speciesHomo sapiens (human)
Saccharomyces cerevisiae BY4743 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsMiller-Vedam, L.E. / Schirle Oakdale, N.S. / Braeuning, B. / Boydston, E.A. / Sevillano, N. / Popova, K.D. / Bonnar, J.L. / Shurtleff, M.J. / Prabu, J.R. / Stroud, R.M. ...Miller-Vedam, L.E. / Schirle Oakdale, N.S. / Braeuning, B. / Boydston, E.A. / Sevillano, N. / Popova, K.D. / Bonnar, J.L. / Shurtleff, M.J. / Prabu, J.R. / Stroud, R.M. / Craik, C.S. / Schulman, B.A. / Weissman, J.S. / Frost, A.
Funding support United States, 2items
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, 2020Deposition site: RCSB / Processing site: RCSB
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 1.2Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-23033
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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: ER membrane protein complex subunit 5
F: ER membrane protein complex subunit 6
G: Protein SOP4
H: Endoplasmic reticulum membrane protein complex subunit 10
I: Fab DH4 heavy chain
J: Fab DH4 light chain
M: Unassigned helix
N: Unassigned helix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,20418
Polymers309,87712
Non-polymers1,3276
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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ER membrane protein complex subunit ... , 6 types, 6 molecules ABCDEF

#1: Protein ER membrane protein complex subunit 1


Mass: 87272.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: P25574
#2: Protein ER membrane protein complex subunit 2


Mass: 33893.211 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: P47133
#3: Protein ER membrane protein complex subunit 3 / Altered inheritance rate of mitochondria protein 27


Mass: 28372.842 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: P36039
#4: Protein ER membrane protein complex subunit 4


Mass: 21478.721 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: P53073
#5: Protein ER membrane protein complex subunit 5 / Killer toxin-resistance protein 27


Mass: 20489.887 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: P40540
#6: Protein ER membrane protein complex subunit 6


Mass: 12411.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: Q12431

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Protein , 2 types, 2 molecules GH

#7: Protein Protein SOP4 / Suppressor of PMA1-7 protein 4


Mass: 26627.627 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: P39543
#8: Protein Endoplasmic reticulum membrane protein complex subunit 10


Mass: 22792.824 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743 / References: UniProt: Q12025

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Antibody , 2 types, 2 molecules IJ

#9: Antibody Fab DH4 heavy chain


Mass: 26849.994 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): BL21-Gold(DE3)pLysS AG
#10: Antibody Fab DH4 light chain


Mass: 25566.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): BL21-Gold(DE3)pLysS AG

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Protein/peptide / Sugars , 2 types, 8 molecules MN

#11: Protein/peptide Unassigned helix


Mass: 2060.531 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4743 (yeast) / Strain: BY4743
#12: 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.: 6 / 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
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1S. cerevisiae endoplasmic reticulum membrane protein complex (EMC) bound to Fab in DDM detergentCOMPLEX#1-#110MULTIPLE SOURCES
2endoplasmic reticulum membrane protein complexCOMPLEX#1-#8, #111NATURAL
3Fab DH4COMPLEX#9-#101RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Saccharomyces cerevisiae BY4743 (yeast)1266529BY4741
23Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: BL21-Gold(DE3)pLysS AG
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company /
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELD / Specimen-ID: 1

IDCryogenDetailsModelSpecimen holder model
1NITROGENDataset 1 collected on FEI Polara at UCSF on August 22nd, 2016.FEI POLARA 300
2Dataset 2 collected on FEI Titan Krios at Janelia Research Campus on October 20th, 2016.FEI TITAN KRIOSFEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1156.8SUPER-RESOLUTIONGATAN K2 SUMMIT (4k x 4k)
2258.3SUPER-RESOLUTIONGATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.1_3865refinement
PHENIX1.18.1_3865refinement
EM software
IDNameVersionCategoryImaging-IDImage processing-ID
2Leginonimage acquisition1
10SerialEMimage acquisition2
11RELIONinitial Euler assignment1
20RELIONinitial Euler assignment2
25PHENIX1.18model refinement
Image processing
IDImage recording-ID
11
22
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction

Entry-ID: 7KTX / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDNum. of particlesImage processing-IDDetails
1835991These 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.
21701862
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 167.41 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005717690
ELECTRON MICROSCOPYf_angle_d0.988424003
ELECTRON MICROSCOPYf_chiral_restr0.05522747
ELECTRON MICROSCOPYf_plane_restr0.00693021
ELECTRON MICROSCOPYf_dihedral_angle_d18.07586402

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