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- PDB-8cr1: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer -

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Basic information

Entry
Database: PDB / ID: 8cr1
TitleHomo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer
Components
  • ATPase ASNA1
  • Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1,GET2-GET1
KeywordsMEMBRANE PROTEIN / membrane protein insertion / GET pathway / tail anchored membrane protein
Function / homology
Function and homology information


arsenite transmembrane transporter activity / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / receptor recycling / protein insertion into ER membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / B cell homeostasis / membrane-membrane adaptor activity / vesicle-mediated transport ...arsenite transmembrane transporter activity / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / receptor recycling / protein insertion into ER membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / B cell homeostasis / membrane-membrane adaptor activity / vesicle-mediated transport / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / negative regulation of protein ubiquitination / epidermal growth factor receptor signaling pathway / defense response / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein stabilization / ubiquitin protein ligase binding / endoplasmic reticulum membrane / nucleolus / endoplasmic reticulum / signal transduction / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / nucleus / membrane / metal ion binding / 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: 3.2 Å
AuthorsMcDowell, M.A. / Heimes, M. / Wild, K. / Sinning, I.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)Leibniz SI 586/6-1 Germany
German Research Foundation (DFG)TRR83 TP22 Germany
Citation
Journal: Nat Commun / Year: 2023
Title: The GET insertase exhibits conformational plasticity and induces membrane thinning.
Authors: Melanie A McDowell / Michael Heimes / Giray Enkavi / Ákos Farkas / Daniel Saar / Klemens Wild / Blanche Schwappach / Ilpo Vattulainen / Irmgard Sinning /
Abstract: The eukaryotic guided entry of tail-anchored proteins (GET) pathway mediates the biogenesis of tail-anchored (TA) membrane proteins at the endoplasmic reticulum. In the cytosol, the Get3 chaperone ...The eukaryotic guided entry of tail-anchored proteins (GET) pathway mediates the biogenesis of tail-anchored (TA) membrane proteins at the endoplasmic reticulum. In the cytosol, the Get3 chaperone captures the TA protein substrate and delivers it to the Get1/Get2 membrane protein complex (GET insertase), which then inserts the substrate via a membrane-embedded hydrophilic groove. Here, we present structures, atomistic simulations and functional data of human and Chaetomium thermophilum Get1/Get2/Get3. The core fold of the GET insertase is conserved throughout eukaryotes, whilst thinning of the lipid bilayer occurs in the vicinity of the hydrophilic groove to presumably lower the energetic barrier of membrane insertion. We show that the gating interaction between Get2 helix α3' and Get3 drives conformational changes in both Get3 and the Get1/Get2 membrane heterotetramer. Thus, we provide a framework to understand the conformational plasticity of the GET insertase and how it remodels its membrane environment to promote substrate insertion.
#1: Journal: 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
DepositionMar 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 13, 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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATPase ASNA1
C: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1,GET2-GET1
B: ATPase ASNA1
D: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1,GET2-GET1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,5245
Polymers150,4594
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry, cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9190 Å2
ΔGint-125 kcal/mol
Surface area60700 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 Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1,GET2-GET1 / Calcium signal-modulating cyclophilin ligand / Congenital heart disease 5 protein / Tail-anchored ...Calcium signal-modulating cyclophilin ligand / Congenital heart disease 5 protein / Tail-anchored protein insertion receptor WRB / Tryptophan-rich basic protein


Mass: 35083.309 Da / Num. of mol.: 2 / Mutation: Truncation of 185 N-terminal residues.
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAMLG, CAML, GET2, GET1, CHD5, WRB / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P49069, UniProt: O00258
#3: 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
IDNameTypeDetailsEntity IDParent-IDSource
1Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimerCOMPLEXGet2-Get1 was expressed as a fusion protein in S. frugiperda and Get3 was expressed in E. coli. The complex components were purified and reconstituted in vitro.#1-#20MULTIPLE SOURCES
2Tail-anchored protein insertion receptor Get1 and Get2/CAMLCOMPLEX#21RECOMBINANT
3Dimeric ATPase Get3COMPLEX#11RECOMBINANT
Molecular weightValue: 0.150 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2200 mMSodium Chloride1
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

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1Warpparticle selection
2SerialEMimage acquisition
4cryoSPARC3.2CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.2initial Euler assignment
11cryoSPARC3.2final Euler assignment
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1561837
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 189844 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039400
ELECTRON MICROSCOPYf_angle_d0.71212712
ELECTRON MICROSCOPYf_dihedral_angle_d3.9831228
ELECTRON MICROSCOPYf_chiral_restr0.0451468
ELECTRON MICROSCOPYf_plane_restr0.0051582

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