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- EMDB-16801: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-16801
TitleHomo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer
Map data
Sample
  • Complex: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer
    • Complex: Tail-anchored protein insertion receptor Get1 and Get2/CAML
      • Protein or peptide: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1,GET2-GET1
    • 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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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: 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, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16801.png

Downloads & links

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Map

FileDownload / File: emd_16801.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 328 pix.
= 265.68 Å		0.81 Å/pix.
x 328 pix.
= 265.68 Å		0.81 Å/pix.
x 328 pix.
= 265.68 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.36
Minimum - Maximum-3.2537925 - 4.1317363
Average (Standard dev.)-0.000070158334 (±0.056902092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 265.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16801_msk_1.map
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Additional map: Final reconstruction low-pass filtered to 6 A resolution

Fileemd_16801_additional_1.map
AnnotationFinal reconstruction low-pass filtered to 6 A resolution
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Half map: #2

Fileemd_16801_half_map_1.map
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Half map: #1

Fileemd_16801_half_map_2.map
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Sample components

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Entire : Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer

EntireName: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer
Components
  • Complex: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer
    • Complex: Tail-anchored protein insertion receptor Get1 and Get2/CAML
      • Protein or peptide: Guided entry of tail-anchored proteins factor CAMLG,Guided entry of tail-anchored proteins factor 1,GET2-GET1
    • Complex: Dimeric ATPase Get3
      • Protein or peptide: ATPase ASNA1
  • Ligand: ZINC ION

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Supramolecule #1: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer

SupramoleculeName: Homo sapiens Get1/Get2 heterotetramer in complex with a Get3 dimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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.
Molecular weightTheoretical: 150 KDa

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Supramolecule #2: Tail-anchored protein insertion receptor Get1 and Get2/CAML

SupramoleculeName: Tail-anchored protein insertion receptor Get1 and Get2/CAML
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 / 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,GET2-GET1
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.083309 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDSFRIFRLV GCALLALGVR AFVCKYLSIF APFLTLQLAY MGLYKYFPKS EKKIKTTVLT AALLLSGIPA EVINRSMDTY SKMGEVFTD LCVYFFTFIF CHELLDYWGS EVPGSGSENL YFQSGSGSMS SAAADHWAWL LVLSFVFGCN VLRILLPSFS S FMSRVLQK ...String:
MDSFRIFRLV GCALLALGVR AFVCKYLSIF APFLTLQLAY MGLYKYFPKS EKKIKTTVLT AALLLSGIPA EVINRSMDTY SKMGEVFTD LCVYFFTFIF CHELLDYWGS EVPGSGSENL YFQSGSGSMS SAAADHWAWL LVLSFVFGCN VLRILLPSFS S FMSRVLQK DAEQESQMRA EIQDMKQELS TVNMMDEFAR YARLERKINK MTDKLKTHVK ARTAQLAKIK WVISVAFYVL QA ALMISLI WKYYSVPVAV VPSKWITPLD RLVAFPTRVA GGVGITCWIL VCNKVVAIVL HPFSGSGSLE VLFQ

UniProtKB: 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

Concentration1.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
200.0 mMSodium Chloride
GridModel: Quantifoil R2/1 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
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 K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 9470 / Average exposure time: 12.0 sec. / Average electron dose: 45.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1561837
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 189844
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

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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