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- PDB-6so5: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer -

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Basic information

Entry
Database: PDB / ID: 6so5
TitleHomo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer
Components
  • ATPase ASNA1
  • Calcium signal-modulating cyclophilin ligand
  • Tail-anchored protein insertion receptor WRB
KeywordsMEMBRANE PROTEIN / ER / insertase / complex / tail-anchor
Function / homology
Function and homology information


arsenite transmembrane transporter activity / otic vesicle development / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / receptor recycling / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / B cell homeostasis ...arsenite transmembrane transporter activity / otic vesicle development / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / receptor recycling / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / B cell homeostasis / vesicle-mediated transport / protein-membrane adaptor activity / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / negative regulation of protein ubiquitination / establishment of localization in cell / sensory perception of sound / defense response / synapse organization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / epidermal growth factor receptor signaling pathway / protein stabilization / ubiquitin protein ligase binding / endoplasmic reticulum membrane / nucleolus / endoplasmic reticulum / signal transduction / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / metal ion binding / nucleus / membrane / cytoplasm
Similarity search - Function
Guided entry of tail-anchored proteins factor CAMLG / Get2-like / Get1 family / CHD5-like protein / Arsenical pump ATPase, ArsA/GET3, eukaryotic / Arsenical pump ATPase, ArsA/GET3 / Anion-transporting ATPase-like domain / Anion-transporting ATPase / Helix hairpin bin domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guided entry of tail-anchored proteins factor 1 / ATPase GET3 / Guided entry of tail-anchored proteins factor CAMLG
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsMcDowell, M.A. / Heimes, M. / Wild, K. / Flemming, D. / Sinning, I.
Funding support Germany, European Union, 3items
OrganizationGrant numberCountry
German Research Foundation (DFG)Leibniz SI 586/6-1 Germany
German Research Foundation (DFG)TRR83 TP22 Germany
European Molecular Biology Organization (EMBO)ALTF 1230-2013European Union
CitationJournal: Mol Cell / Year: 2020
Title: Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex.
Authors: Melanie A McDowell / Michael Heimes / Francesco Fiorentino / Shahid Mehmood / Ákos Farkas / Javier Coy-Vergara / Di Wu / Jani Reddy Bolla / Volker Schmid / Roger Heinze / Klemens Wild / ...Authors: Melanie A McDowell / Michael Heimes / Francesco Fiorentino / Shahid Mehmood / Ákos Farkas / Javier Coy-Vergara / Di Wu / Jani Reddy Bolla / Volker Schmid / Roger Heinze / Klemens Wild / Dirk Flemming / Stefan Pfeffer / Blanche Schwappach / Carol V Robinson / Irmgard Sinning /
Abstract: Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and ...Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.
History
DepositionAug 29, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 9, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2021Group: Database references / Category: citation
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.page_first / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 10, 2024Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_audit_support / pdbx_database_related / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_audit_support.country
Revision 1.3Nov 20, 2024Group: Data collection / Structure summary
Category: 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

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Structure viewerMolecule:
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Assembly

Deposited unit
A: ATPase ASNA1
B: ATPase ASNA1
C: Tail-anchored protein insertion receptor WRB
D: Tail-anchored protein insertion receptor WRB
E: Calcium signal-modulating cyclophilin ligand
F: Calcium signal-modulating cyclophilin ligand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,5607
Polymers150,4956
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10160 Å2
ΔGint-139 kcal/mol
Surface area61640 Å2
MethodPISA

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Components

#1: Protein ATPase ASNA1 / Arsenical pump-driving ATPase / Arsenite-stimulated ATPase / Transmembrane domain recognition ...Arsenical pump-driving ATPase / Arsenite-stimulated ATPase / Transmembrane domain recognition complex 40 kDa ATPase subunit / hARSA-I / hASNA-I


Mass: 40146.070 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ASNA1, ARSA, TRC40 / Production host: Escherichia coli (E. coli)
References: UniProt: O43681, Hydrolases; Acting on acid anhydrides
#2: Protein Tail-anchored protein insertion receptor WRB / Congenital heart disease 5 protein / Tryptophan-rich basic protein / WRB


Mass: 20821.668 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WRB, CHD5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O00258
#3: Protein Calcium signal-modulating cyclophilin ligand / CAML


Mass: 14279.671 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAMLG, CAML / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P49069
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
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
1Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimerCOMPLEX#1-#30MULTIPLE SOURCES
2ATPase ASNA1COMPLEX#11RECOMBINANT
3Tail-anchored protein insertion receptor WRB and calcium signal-modulating cyclophilin ligandCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.150 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2200 mMNaCl1
SpecimenConc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: complex stabilised in PMAL-C8 amphipol
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12 sec. / Electron dose: 45.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9470
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategoryFitting-ID
1Warpparticle selection
2SerialEMimage acquisition
4RELION3CTF correction
7Cootmodel fitting1
13RELION3classification
15RELION33D reconstruction
33Cootmodel fitting2
40PHENIXmodel refinement2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1698096
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225244 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolDetailsSpace
1RIGID BODY FIT3SJA
2BACKBONE TRACEREAL
Atomic model buildingPDB-ID: 3SJA

3sja


Accession code: 3SJA / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058918
ELECTRON MICROSCOPYf_angle_d0.75212045
ELECTRON MICROSCOPYf_dihedral_angle_d5.3845406
ELECTRON MICROSCOPYf_chiral_restr0.0451386
ELECTRON MICROSCOPYf_plane_restr0.0051497

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