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- EMDB-16802: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in c... -

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Basic information

Entry
Database: EMDB / ID: EMD-16802
TitleHomo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in complex with a Get3 dimer
Map data
Sample
  • Complex: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in complex with a Get3 dimer
    • Complex: Tail-anchored protein insertion receptor Get1 and Get2/CAML (a3' deletion variant)
      • Protein or peptide: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1
    • Complex: Dimeric ATPase Get3
      • Protein or peptide: ATPase ASNA1
  • Ligand: ZINC ION
Keywordsmembrane protein insertion / GET pathway / tail anchored membrane protein / 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 / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / B cell homeostasis / protein-membrane adaptor activity ...arsenite transmembrane transporter activity / membrane insertase activity / GET complex / tail-anchored membrane protein insertion into ER membrane / receptor recycling / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / B cell homeostasis / protein-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 / protein stabilization / ubiquitin protein ligase binding / endoplasmic reticulum membrane / nucleolus / endoplasmic reticulum / signal transduction / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / membrane / nucleus / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsMcDowell MA / Heimes M / Wild K / Sinning I
Funding support Germany, 2 items
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, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16802.png

Downloads & links

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Map

FileDownload / File: emd_16802.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 256 pix.
= 284.16 Å		1.11 Å/pix.
x 256 pix.
= 284.16 Å		1.11 Å/pix.
x 256 pix.
= 284.16 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.11 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.21
Minimum - Maximum-1.3963249 - 2.02653
Average (Standard dev.)-0.000121417645 (±0.028358068)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 284.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16802_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Half map: #2

Fileemd_16802_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_16802_half_map_2.map
Projections & Slices
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Sample components

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Entire : Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in c...

EntireName: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in complex with a Get3 dimer
Components
  • Complex: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in complex with a Get3 dimer
    • Complex: Tail-anchored protein insertion receptor Get1 and Get2/CAML (a3' deletion variant)
      • Protein or peptide: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1
    • Complex: Dimeric ATPase Get3
      • Protein or peptide: ATPase ASNA1
  • Ligand: ZINC ION

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Supramolecule #1: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in c...

SupramoleculeName: Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in complex with a Get3 dimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Get2-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.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Tail-anchored protein insertion receptor Get1 and Get2/CAML (a3' ...

SupramoleculeName: Tail-anchored protein insertion receptor Get1 and Get2/CAML (a3' deletion variant)
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Dimeric ATPase Get3

SupramoleculeName: Dimeric ATPase Get3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: ATPase ASNA1

MacromoleculeName: ATPase ASNA1 / type: protein_or_peptide / ID: 1 / Details: Get3 was expressed in E. coli / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on acid anhydrides
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.14607 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMAAGVAGW GVEAEEFEDA PDVEPLEPTL SNIIEQRSLK WIFVGGKGGV GKTTCSCSLA VQLSKGRESV LIISTDPAHN ISDAFDQKF SKVPTKVKGY DNLFAMEIDP SLGVAELPDE FFEEDNMLSM GKKMMQEAMS AFPGIDEAMS YAEVMRLVKG M NFSVVVFD ...String:
GAMAAGVAGW GVEAEEFEDA PDVEPLEPTL SNIIEQRSLK WIFVGGKGGV GKTTCSCSLA VQLSKGRESV LIISTDPAHN ISDAFDQKF SKVPTKVKGY DNLFAMEIDP SLGVAELPDE FFEEDNMLSM GKKMMQEAMS AFPGIDEAMS YAEVMRLVKG M NFSVVVFD TAPTGHTLRL LNFPTIVERG LGRLMQIKNQ ISPFISQMCN MLGLGDMNAD QLASKLEETL PVIRSVSEQF KD PEQTTFI CVCIAEFLSL YETERLIQEL AKCKIDTHNI IVNQLVFPDP EKPCKMCEAR HKIQAKYLDQ MEDLYEDFHI VKL PLLPHE VRGADKVNTF SALLLEPYKP PSAQGSWSHP QFEK

UniProtKB: ATPase GET3

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Macromolecule #2: Guided entry of tail-anchored proteins factor CAMLG,Guided entry ...

MacromoleculeName: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1
type: protein_or_peptide / ID: 2
Details: Get2-Get1 was expressed as a fusion protein in S. frugiperda
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.429418 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDSFRIFRLV GCALLALGVR AFVCKYLSIF APFLTLQLAY MGLYKYFPKS EKKIKTTGGG GGIPAEVINR SMDTYSKMGE VFTDLCVYF FTFIFCHELL DYWGSEVPGS GSENLYFQSG SGSMSSAAAD HWAWLLVLSF VFGCNVLRIL LPSFSSFMSR V LQKDAEQE ...String:
MDSFRIFRLV GCALLALGVR AFVCKYLSIF APFLTLQLAY MGLYKYFPKS EKKIKTTGGG GGIPAEVINR SMDTYSKMGE VFTDLCVYF FTFIFCHELL DYWGSEVPGS GSENLYFQSG SGSMSSAAAD HWAWLLVLSF VFGCNVLRIL LPSFSSFMSR V LQKDAEQE SQMRAEIQDM KQELSTVNMM DEFARYARLE RKINKMTDKL KTHVKARTAQ LAKIKWVISV AFYVLQAALM IS LIWKYYS VPVAVVPSKW ITPLDRLVAF PTRVAGGVGI TCWILVCNKV VAIVLHPFSG SGSLEVLFQ

UniProtKB: Guided entry of tail-anchored proteins factor CAMLG, Guided entry of tail-anchored proteins factor CAMLG, Guided entry of tail-anchored proteins factor 1

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
200.0 mMSodium Chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
DetailsComplex stabilised in PMAL-C8 amphipol

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 12311 / Average exposure time: 2.58 sec. / Average electron dose: 53.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2044854
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 224354
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2) / Details: ab initio reconstruction in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2) / Details: non-uniform refinement in cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6so5


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8cr2:
Homo sapiens Get1/Get2 heterotetramer (a3' deletion variant) in complex with a Get3 dimer

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