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- EMDB-11607: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with... -

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Basic information

Entry
Database: EMDB / ID: EMD-11607
TitleSaccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer
Map data
Sample
  • Complex: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer
    • Complex: ATPase Get3
      • Protein or peptide: Get3
    • Complex: Get1 and Get2
      • Protein or peptide: Get1
      • Protein or peptide: Get2
Function / homology
Function and homology information


establishment of protein localization to endoplasmic reticulum membrane / GET complex / pheromone-dependent signal transduction involved in conjugation with cellular fusion / 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 / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / response to arsenic-containing substance / response to metal ion ...establishment of protein localization to endoplasmic reticulum membrane / GET complex / pheromone-dependent signal transduction involved in conjugation with cellular fusion / 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 / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / response to arsenic-containing substance / response to metal ion / protein transmembrane transporter activity / mitophagy / protein-membrane adaptor activity / protein folding chaperone / guanyl-nucleotide exchange factor activity / mitochondrial membrane / unfolded protein binding / response to heat / cellular response to oxidative stress / Golgi membrane / endoplasmic reticulum membrane / endoplasmic reticulum / Golgi apparatus / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / cytosol
Similarity search - Function
Golgi to ER traffic protein 2 / GET complex subunit Get2/sif1 / GET complex subunit GET2 / Get 1, fungi / Get1 family / CHD5-like protein / Arsenical pump ATPase, ArsA/GET3, eukaryotic / Arsenical pump ATPase, ArsA/GET3 / Anion-transporting ATPase-like domain / Anion-transporting ATPase ...Golgi to ER traffic protein 2 / GET complex subunit Get2/sif1 / GET complex subunit GET2 / Get 1, fungi / 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
Golgi to ER traffic protein 2 / Golgi to ER traffic protein 1 / ATPase GET3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsMcDowell MA / Pfeffer S / Flemming D / Sinning I
Funding support Germany, 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 1230-2013 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
DepositionAug 13, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateMar 3, 2021-
Current statusMar 3, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11607.png

Downloads & links

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Map

FileDownload / File: emd_11607.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.14 Å/pix.
x 96 pix.
= 205.44 Å		2.14 Å/pix.
x 96 pix.
= 205.44 Å		2.14 Å/pix.
x 96 pix.
= 205.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.012029174 - 0.07
Average (Standard dev.)-0.00033695844 (±0.006633482)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 205.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.142.142.14
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z205.440205.440205.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.0120.070-0.000

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with...

EntireName: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer
Components
  • Complex: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer
    • Complex: ATPase Get3
      • Protein or peptide: Get3
    • Complex: Get1 and Get2
      • Protein or peptide: Get1
      • Protein or peptide: Get2

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Supramolecule #1: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with...

SupramoleculeName: Saccharomyces cerevisiae Get1/Get2 heterotetramer in complex with a Get3 dimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 174 KDa

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Supramolecule #2: ATPase Get3

SupramoleculeName: ATPase Get3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Get1 and Get2

SupramoleculeName: Get1 and Get2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Get1

MacromoleculeName: Get1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MHWAAAVAIF FIVVTKFLQY TNK YHEKWI SKFAPGNELS KKYLAKVKER HELKEFNNSI SAQDNYAKWT KNNRKLDSLD KEIN NLKDE IQSENKAFQA HLHKLRLLAL TVPFFVFKIM YGKTPVYKLS SSTSTLFPTF VSGVW SQGW LYVLLHPLRT ISQKWHIMEG ...String:
MHWAAAVAIF FIVVTKFLQY TNK YHEKWI SKFAPGNELS KKYLAKVKER HELKEFNNSI SAQDNYAKWT KNNRKLDSLD KEIN NLKDE IQSENKAFQA HLHKLRLLAL TVPFFVFKIM YGKTPVYKLS SSTSTLFPTF VSGVW SQGW LYVLLHPLRT ISQKWHIMEG KFGASKFDDM ALQSVSLGIW VWALMNVING VEFIVK QLF LTPKMEAPAS VETQEEKA L DAVDDAIILD GSLEVLFQ

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Macromolecule #2: Get2

MacromoleculeName: Get2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MDYHDYLLNR LKAWTILVKW VFFLLPYLYL ITRPN SSVW PAYAFTQSAW FAPLRNPSNF TRIFATFEFL SISIYYQLLK NVEHKSKIKN LQDTNK LVK LVSLVPEGVI PVANLKGKLI T LLQYWD L LSMLITDISF VLIVLGLLTY LGSGSGSGSG SGSGS

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Macromolecule #3: Get3

MacromoleculeName: Get3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDLTVEPNLH SLITSTTHK W IFVGGKGG VG KTTSSCS IAI QMALSQ PNKQ FLLIS TDPAH NLSD AFGEKF GKD ARKVTGM NN LSCMEIDP S AALKDMNDM AVSRANNNGS DGQGDDLGS L LQGGALAD LT GSIPGID EAL SFMEVM KHIK RQEQG ...String:
MDLTVEPNLH SLITSTTHK W IFVGGKGG VG KTTSSCS IAI QMALSQ PNKQ FLLIS TDPAH NLSD AFGEKF GKD ARKVTGM NN LSCMEIDP S AALKDMNDM AVSRANNNGS DGQGDDLGS L LQGGALAD LT GSIPGID EAL SFMEVM KHIK RQEQG EGETF DTVI FDTAPT GHT LRFLQLP NT LSKLLEKF G EITNKLGPM LNSFMGAGNV DISGKLNEL K ANVETIRQ QF TDPDLTT FVC VCISEF LSLY ETERL IQELI SYDM DVNSII VNQ LLFAEND QE HNCKRCQA R WKMQKKYLD QIDELYEDFH VVKMPLCAG E IRGLNNLT KF SQFLNKE YNP ITDGKV IYEL EDKEW SHPQF EK

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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

Concentration1.3 mg/mL
BufferpH: 7.5 / Component:
ConcentrationFormula
20.0 mMHEPES
200.0 mMNaCl
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
Detailscomplex stabilised in A835 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: 678 / Average exposure time: 4.0 sec. / Average electron dose: 25.0 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: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 150857
CTF correctionSoftware - Name: Gctf
Final reconstructionResolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.2) / Number images used: 27666
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial modelPDB ID:

3sja

RefinementProtocol: RIGID BODY FIT

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