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- PDB-8cr2: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in c... -

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Basic information

Entry
Database: PDB / ID: 8cr2
TitleHomo sapiens Get1/Get2 heterotetramer (a3' deletion variant) 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
KeywordsMEMBRANE PROTEIN / membrane protein insertion / GET pathway / tail anchored membrane protein
Function / homology
Function and homology information


arsenite transmembrane transporter activity / receptor recycling / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / Hydrolases; Acting on acid anhydrides / 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 / receptor recycling / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / Hydrolases; Acting on acid anhydrides / 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 / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / protein-membrane adaptor activity / negative regulation of protein ubiquitination / vesicle-mediated transport / defense response / 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 / membrane / metal ion binding / nucleus / 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. / 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 7, 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)149,2165
Polymers149,1514
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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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
Details: Get3 was expressed in E. coli / 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 / 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: 34429.418 Da / Num. of mol.: 2
Mutation: Truncation of 185 N-terminal residues. Residues 242-250 replaced with a GGGG linker
Source method: isolated from a genetically manipulated source
Details: Get2-Get1 was expressed as a fusion protein in S. frugiperda
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

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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 (a3' deletion variant) 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.#10MULTIPLE SOURCES
2Tail-anchored protein insertion receptor Get1 and Get2/CAML (a3' deletion variant)COMPLEX#21RECOMBINANT
3Dimeric ATPase Get3COMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.149 MDaNO
22
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
52Homo sapiens (human)9606
43Homo 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.: 1.2 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 / 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: 2.58 sec. / Electron dose: 53.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12311

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / 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: 2044854
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224354 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6SO5

6so5


Accession code: 6SO5 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047108
ELECTRON MICROSCOPYf_angle_d1.0629593
ELECTRON MICROSCOPYf_dihedral_angle_d4.519927
ELECTRON MICROSCOPYf_chiral_restr0.0571104
ELECTRON MICROSCOPYf_plane_restr0.0071194

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