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- PDB-8cqz: Homo sapiens Get3 in complex with the Get1 cytoplasmic domain -

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Basic information

Entry
Database: PDB / ID: 8cqz
TitleHomo sapiens Get3 in complex with the Get1 cytoplasmic domain
Components
  • ATPase ASNA1
  • Guided entry of tail-anchored proteins factor 1
KeywordsPROTEIN TRANSPORT / 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 / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / protein-membrane adaptor activity / protein stabilization / endoplasmic reticulum membrane ...arsenite transmembrane transporter activity / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / protein-membrane adaptor activity / protein stabilization / endoplasmic reticulum membrane / nucleolus / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
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
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsMcDowell, M.A. / Heimes, M. / Wild, K. / Saar, D. / 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

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Assembly

Deposited unit
A: ATPase ASNA1
B: Guided entry of tail-anchored proteins factor 1


Theoretical massNumber of molelcules
Total (without water)48,3752
Polymers48,3752
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1170 Å2
ΔGint-2 kcal/mol
Surface area23430 Å2
Unit cell
Length a, b, c (Å)69.478, 81.549, 92.185
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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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.: 1
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 1 / Congenital heart disease 5 protein / Tail-anchored protein insertion receptor WRB / Tryptophan-rich ...Congenital heart disease 5 protein / Tail-anchored protein insertion receptor WRB / Tryptophan-rich basic protein


Mass: 8228.452 Da / Num. of mol.: 1 / Mutation: Residues 38-94 of Get1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GET1, CHD5, WRB / Production host: Escherichia coli (E. coli) / References: UniProt: O00258

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.7 Å3/Da / Density % sol: 54.43 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: 44 % (v/v) 1,2-propanediol, 0.05 M calcium acetate and 0.1 M sodium acetate (pH 5.0)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.972 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 18, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.972 Å / Relative weight: 1
ReflectionResolution: 2.8→45.9 Å / Num. obs: 13414 / % possible obs: 96.5 % / Redundancy: 12.8 % / CC1/2: 0.999 / Rmerge(I) obs: 0.045 / Rpim(I) all: 0.018 / Net I/σ(I): 25
Reflection shellResolution: 2.8→2.9 Å / Rmerge(I) obs: 1.579 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 1324 / CC1/2: 0.735 / Rpim(I) all: 0.448

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Processing

Software
NameVersionClassification
PHENIXv1.15refinement
XDSdata reduction
Aimlessdata scaling
STARANISOdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.8→45.9 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / Phase error: 35.76 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2832 1292 9.98 %
Rwork0.2424 --
obs0.2466 12947 96.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.8→45.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3169 0 0 0 3169
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0023228
X-RAY DIFFRACTIONf_angle_d0.4724351
X-RAY DIFFRACTIONf_dihedral_angle_d12.1351240
X-RAY DIFFRACTIONf_chiral_restr0.04488
X-RAY DIFFRACTIONf_plane_restr0.004565
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8-2.910.38571200.34321112X-RAY DIFFRACTION84
2.91-3.040.52511320.39851192X-RAY DIFFRACTION91
3.04-3.20.43141420.35141257X-RAY DIFFRACTION96
3.21-3.410.36551440.29511302X-RAY DIFFRACTION98
3.41-3.670.35781440.30561321X-RAY DIFFRACTION99
3.67-4.040.32811480.2711327X-RAY DIFFRACTION100
4.04-4.620.30431500.23351349X-RAY DIFFRACTION100
4.62-5.810.24811520.23931360X-RAY DIFFRACTION100
5.82-45.90.24291600.20911436X-RAY DIFFRACTION100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
14.801-1.6474-1.41167.27320.03380.2018-0.10010.21080.85471.1006-0.0592-1.24670.38280.1153-0.05931.30630.0598-0.28841.0263-0.23380.383844.940332.331612.5588
22.466-1.3753.17972.2401-0.60735.46980.10831.25541.0531-0.6831-0.2395-0.8182-0.42270.71070.09941.50930.08810.29261.81740.18760.91132.970646.1479-8.8389
37.85591.36-3.15622.2781-1.52315.63590.1961-1.17681.15150.98560.2549-0.51610.32030.0361-0.42451.53920.2112-0.32741.4453-0.35420.616931.865737.400822.3802
45.2579-0.60090.80599.53256.89575.22890.4153-0.3897-0.8892.3743-1.04520.48272.96490.15690.57991.9296-0.0589-0.24771.3264-0.14810.792722.15825.51714.7411
53.29250.5532.5578.99562.37036.70560.3271.7065-0.8184-0.7775-0.0735-0.98882.1099-0.1957-0.04621.5504-0.07190.00451.5485-0.22730.692524.046514.16260.449
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 7 through 64 )
2X-RAY DIFFRACTION2chain 'A' and (resid 65 through 251 )
3X-RAY DIFFRACTION3chain 'A' and (resid 252 through 347 )
4X-RAY DIFFRACTION4chain 'B' and (resid 39 through 62 )
5X-RAY DIFFRACTION5chain 'B' and (resid 63 through 104 )

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