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- PDB-8odv: Chaetomium thermophilum Get1/Get2 heterotetramer in complex with ... -

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Basic information

Entry
Database: PDB / ID: 8odv
TitleChaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimer (nanodisc)
Components
  • ATPase GET3
  • Protein GET2,Protein GET1
KeywordsMEMBRANE PROTEIN / membrane protein insertion / GET pathway / tail anchored membrane protein
Function / homology
Function and homology information


GET complex / tail-anchored membrane protein insertion into ER membrane / protein insertion into ER membrane / endoplasmic reticulum membrane / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / membrane / metal ion binding
Similarity search - Function
GET complex subunit Get2/sif1 / GET complex subunit GET2 / Get 1, fungi / 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
Uncharacterized protein / Protein GET1 / Arsenite translocating ATPase-like protein
Similarity search - Component
Biological speciesThermochaetoides thermophila DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsMcDowell, M.A. / 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
CitationJournal: 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.
History
DepositionMar 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATPase GET3
B: ATPase GET3
C: Protein GET2,Protein GET1
D: Protein GET2,Protein GET1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,4685
Polymers165,4024
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6130 Å2
ΔGint-64 kcal/mol
Surface area63450 Å2
MethodPISA

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Components

#1: Protein ATPase GET3 / Arsenical pump-driving ATPase / Arsenite-stimulated ATPase / Golgi to ER traffic protein 3 / Guided ...Arsenical pump-driving ATPase / Arsenite-stimulated ATPase / Golgi to ER traffic protein 3 / Guided entry of tail-anchored proteins 3


Mass: 37127.391 Da / Num. of mol.: 2 / Mutation: Truncation of 13 N-terminal residues
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermochaetoides thermophila DSM 1495 (fungus)
Gene: GET3, CTHT_0061710 / Production host: Escherichia coli (E. coli)
References: UniProt: G0SFE0, Hydrolases; Acting on acid anhydrides
#2: Protein Protein GET2,Protein GET1 / Guided entry of tail-anchored proteins factor CAMLG / CAMLG / CAML / GET2 / Guided entry of tail- ...Guided entry of tail-anchored proteins factor CAMLG / CAMLG / CAML / GET2 / Guided entry of tail-anchored proteins 1 / GET1


Mass: 45573.727 Da / Num. of mol.: 2 / Mutation: Truncation of 184 N-terminal residues.
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermochaetoides thermophila DSM 1495 (fungus)
Gene: CTHT_0002670, GET1, CTHT_0013590 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: G0RZE4, UniProt: G0S1H2
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Chaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimerCOMPLEXGet2-Get1 was expressed as a fusion protein in S. cerevisiae and Get3 was expressed in E. coli. The complex components were purified and reconstituted in vitro.#1-#20MULTIPLE SOURCES
2Tail-anchored protein insertase Get1 and Get2COMPLEX#21RECOMBINANT
3Dimeric ATPase Get3COMPLEX#11RECOMBINANT
Molecular weightValue: 0.165 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Thermochaetoides thermophila (fungus)209285
33Thermochaetoides thermophila (fungus)209285
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Saccharomyces cerevisiae (brewer's yeast)4932
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMSodium Chloride1
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Complex stabilised in a Msp1E3D1 bound nanodisc
Specimen supportGrid material: COPPER / 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 FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 60.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11997

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1Warpparticle selection
2EPUimage acquisition
4cryoSPARC3.2CTF correction
7Cootmodel fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
12cryoSPARC3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5218800
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 259692 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0078412
ELECTRON MICROSCOPYf_angle_d1.09511384
ELECTRON MICROSCOPYf_dihedral_angle_d8.2865050
ELECTRON MICROSCOPYf_chiral_restr0.0521286
ELECTRON MICROSCOPYf_plane_restr0.0081426

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