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- EMDB-10266: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-10266
TitleHomo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer
Map data
Sample
  • Complex: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer
    • Complex: ATPase ASNA1
      • Protein or peptide: ATPase ASNA1
    • Complex: Tail-anchored protein insertion receptor WRB and calcium signal-modulating cyclophilin ligand
      • Protein or peptide: Tail-anchored protein insertion receptor WRB
      • Protein or peptide: Calcium signal-modulating cyclophilin ligand
  • Ligand: ZINC ION
KeywordsER / insertase / complex / tail-anchor / 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 / protein-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 / protein-membrane adaptor activity / vesicle-mediated transport / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / negative regulation of protein ubiquitination / defense response / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / 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 / metal ion binding / nucleus / membrane / 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
Funding support Germany, European Union, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)Leibniz SI 586/6-1 Germany
German Research Foundation (DFG)TRR83 TP22 Germany
European Molecular Biology Organization (EMBO)ALTF 1230-2013European Union
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
DepositionAug 29, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 9, 2020-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6so5
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6so5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10266.png

Downloads & links

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Map

FileDownload / File: emd_10266.map.gz / Format: CCP4 / Size: 91.1 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 288 pix.
= 233.28 Å		0.81 Å/pix.
x 288 pix.
= 233.28 Å		0.81 Å/pix.
x 288 pix.
= 233.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.037962444 - 0.057236042
Average (Standard dev.)0.00016846736 (±0.0021505535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 233.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.810.810.81
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z233.280233.280233.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0380.0570.000

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Supplemental data

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Mask #1

Fileemd_10266_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_10266_additional.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer

EntireName: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer
Components
  • Complex: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer
    • Complex: ATPase ASNA1
      • Protein or peptide: ATPase ASNA1
    • Complex: Tail-anchored protein insertion receptor WRB and calcium signal-modulating cyclophilin ligand
      • Protein or peptide: Tail-anchored protein insertion receptor WRB
      • Protein or peptide: Calcium signal-modulating cyclophilin ligand
  • Ligand: ZINC ION

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Supramolecule #1: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer

SupramoleculeName: Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 150 KDa

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Supramolecule #2: ATPase ASNA1

SupramoleculeName: ATPase ASNA1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Tail-anchored protein insertion receptor WRB and calcium signal-m...

SupramoleculeName: Tail-anchored protein insertion receptor WRB and calcium signal-modulating cyclophilin ligand
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
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: Tail-anchored protein insertion receptor WRB

MacromoleculeName: Tail-anchored protein insertion receptor WRB / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.821668 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MSSAAADHWA WLLVLSFVFG CNVLRILLPS FSSFMSRVLQ KDAEQESQMR AEIQDMKQEL STVNMMDEFA RYARLERKIN KMTDKLKTH VKARTAQLAK IKWVISVAFY VLQAALMISL IWKYYSVPVA VVPSKWITPL DRLVAFPTRV AGGVGITCWI L VCNKVVAI VLHPFSGSGS LEVLFQ

UniProtKB: Guided entry of tail-anchored proteins factor 1

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Macromolecule #3: Calcium signal-modulating cyclophilin ligand

MacromoleculeName: Calcium signal-modulating cyclophilin ligand / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.279671 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MDSFRIFRLV GCALLALGVR AFVCKYLSIF APFLTLQLAY MGLYKYFPKS EKKIKTTVLT AALLLSGIPA EVINRSMDTY SKMGEVFTD LCVYFFTFIF CHELLDYWGS EVPGSGSENL YFQSGSGS

UniProtKB: Guided entry of tail-anchored proteins factor CAMLG

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / 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:
ConcentrationFormula
20.0 mMHEPES
200.0 mMNaCl
GridModel: Quantifoil R2/1 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 2 sec.
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: 1698096
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 225244
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3sja


Chain - Source name: PDB / Chain - Initial model type: experimental model
Details3SJA
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6so5:
Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer

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Atomic model buiding 2

RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-6so5:
Homo sapiens WRB/CAML heterotetramer in complex with a TRC40 dimer

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