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- PDB-8s9s: Structure of the human ER membrane protein complex (EMC) in GDN -

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Basic information

Entry
Database: PDB / ID: 8s9s
TitleStructure of the human ER membrane protein complex (EMC) in GDN
Components
  • (ER membrane protein complex subunit ...) x 8
  • Membrane magnesium transporter 1
KeywordsMEMBRANE PROTEIN / Insertase / endoplasmic reticulum / transmembrane chaperone
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 / tail-anchored membrane protein insertion into ER membrane / Miscellaneous transport and binding events / ferrous iron 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 / tail-anchored membrane protein insertion into ER membrane / Miscellaneous transport and binding events / ferrous iron transmembrane transporter activity / magnesium ion 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 / endoplasmic reticulum membrane / apoptotic process / 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) / EMC2 TPR-like repeat domain / 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 8/9 / Uncharacterised protein family (UPF0172) / EMC2 TPR-like repeat domain / 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 / Carbohydrate-binding-like fold / Pyrrolo-quinoline quinone repeat / PQQ-like domain / 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: 3.6 Å
AuthorsTomaleri, G.P. / Nguyen, V.N. / Voorhees, R.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM137412 United States
CitationJournal: J Cell Biol / Year: 2023
Title: A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER.
Authors: Tino Pleiner / Masami Hazu / Giovani Pinton Tomaleri / Vy N Nguyen / Kurt Januszyk / Rebecca M Voorhees /
Abstract: Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA ...Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA proteins to the ER, where they are delivered to the insertase, the ER membrane protein complex (EMC). Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA protein from its cytosolic capture by methionine-rich loops to its membrane insertion through a hydrophilic vestibule. Positively charged residues at the entrance to the vestibule function as a selectivity filter that uses charge-repulsion to reject mitochondrial TA proteins. Similarly, this selectivity filter retains the positively charged soluble domains of multipass substrates in the cytosol, thereby ensuring they adopt the correct topology and enforcing the "positive-inside" rule. Substrate discrimination by the EMC provides a biochemical explanation for one role of charge in TA protein sorting and protects compartment integrity by limiting protein misinsertion.
History
DepositionMar 30, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1May 31, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 14, 2023Group: Structure summary / Category: audit_author / Item: _audit_author.name
Revision 1.3Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: ER membrane protein complex subunit 1
2: ER membrane protein complex subunit 2
3: ER membrane protein complex subunit 3
4: ER membrane protein complex subunit 4
5: Membrane magnesium transporter 1
6: ER membrane protein complex subunit 6
7: ER membrane protein complex subunit 7
8: ER membrane protein complex subunit 8
10: ER membrane protein complex subunit 10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)307,49319
Polymers301,2769
Non-polymers6,21710
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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ER membrane protein complex subunit ... , 8 types, 8 molecules 123467810

#1: Protein ER membrane protein complex subunit 1


Mass: 111886.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) 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 natural source / Source: (natural) 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 natural source / Source: (natural) 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 natural source / Source: (natural) 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 natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BV81
#7: Protein ER membrane protein complex subunit 7


Mass: 26501.586 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9NPA0
#8: 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 natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43402
#9: Protein ER membrane protein complex subunit 10 / Hematopoietic signal peptide-containing membrane domain-containing protein 1


Mass: 27375.797 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q5UCC4

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Protein / Non-polymers , 2 types, 7 molecules 5

#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.: 6 / 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: 14706.786 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q8N4V1

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Sugars , 2 types, 4 molecules

#10: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / 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

ComponentName: Human ER Membrane Protein Complex / Type: COMPLEX / Entity ID: #1-#9 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acidHEPES1
2200 mMSodium chlorideNaCl1
32 mMmagnesium acetateMgAc21
41 mMDL-DithiothreitoDTT1
5.05 % (w/v)Synthetic substitute for DigitoninGDN1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample solubilized and purified in GDN
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: DARK FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.66 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11822
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3particle selection
2SerialEMimage acquisition
4cryoSPARC3.3CTF correction
7UCSF Chimera1.4model fitting
10cryoSPARC3.3final Euler assignment
11cryoSPARC4classification
12cryoSPARC43D reconstruction
13PHENIX1.20.1-4487model refinementphenix.real_space_refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1034250
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 156706 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003218404
ELECTRON MICROSCOPYf_angle_d0.619824950
ELECTRON MICROSCOPYf_chiral_restr0.04082805
ELECTRON MICROSCOPYf_plane_restr0.00493159
ELECTRON MICROSCOPYf_dihedral_angle_d5.63972597

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