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- EMDB-16819: Chaetomium thermophilum Get1/Get2 heterotetramer in complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-16819
TitleChaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimer (nanodisc)
Map data
Sample
  • Complex: Chaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimer
    • Complex: Tail-anchored protein insertase Get1 and Get2
      • Protein or peptide: Protein GET2,Protein GET1
    • Complex: Dimeric ATPase Get3
      • Protein or peptide: ATPase GET3
  • Ligand: ZINC ION
Keywordsmembrane protein insertion / GET pathway / tail anchored membrane protein / MEMBRANE PROTEIN
Function / homology
Function and homology information


GET complex / tail-anchored membrane protein insertion into ER membrane / protein insertion into ER membrane / endoplasmic reticulum membrane / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / membrane / metal ion binding
Similarity search - Function
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
Uncharacterized protein / Protein GET1 / Arsenite translocating ATPase-like protein
Similarity search - Component
Biological speciesThermochaetoides thermophila (fungus) / Thermochaetoides thermophila DSM 1495 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsMcDowell MA / 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 9, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16819.png

Downloads & links

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Map

FileDownload / File: emd_16819.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: 0.138
Minimum - Maximum-0.4168781 - 0.78497994
Average (Standard dev.)0.0004632273 (±0.0208062)
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_16819_msk_1.map
Projections & Slices
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Additional map: Final map low-pass filtered to 6.5 A

Fileemd_16819_additional_1.map
AnnotationFinal map low-pass filtered to 6.5 A
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Half map: #2

Fileemd_16819_half_map_1.map
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Half map: #1

Fileemd_16819_half_map_2.map
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Sample components

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Entire : Chaetomium thermophilum Get1/Get2 heterotetramer in complex with ...

EntireName: Chaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimer
Components
  • Complex: Chaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimer
    • Complex: Tail-anchored protein insertase Get1 and Get2
      • Protein or peptide: Protein GET2,Protein GET1
    • Complex: Dimeric ATPase Get3
      • Protein or peptide: ATPase GET3
  • Ligand: ZINC ION

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Supramolecule #1: Chaetomium thermophilum Get1/Get2 heterotetramer in complex with ...

SupramoleculeName: Chaetomium thermophilum 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. cerevisiae and Get3 was expressed in E. coli. The complex components were purified and reconstituted in vitro.
Molecular weightTheoretical: 165 KDa

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Supramolecule #2: Tail-anchored protein insertase Get1 and Get2

SupramoleculeName: Tail-anchored protein insertase Get1 and Get2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Thermochaetoides thermophila (fungus)

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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: Thermochaetoides thermophila (fungus)

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Macromolecule #1: ATPase GET3

MacromoleculeName: ATPase GET3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on acid anhydrides
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 37.127391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEPTLQSILD QRSLRWIFVG GKGGVGKTTT SCSLAIQLAK VRRSVLLLST DPAHNLSDAF SQKFGKEARL VEGFDNLYAM EIDPNGSMQ DLLAGQTGDG DAGMGGVGVM QDLAYAIPGI DEAMSFAEVL KQVNSLSYET IVFDTAPTGH TLRFLQFPTV L EKALAKVS ...String:
MEPTLQSILD QRSLRWIFVG GKGGVGKTTT SCSLAIQLAK VRRSVLLLST DPAHNLSDAF SQKFGKEARL VEGFDNLYAM EIDPNGSMQ DLLAGQTGDG DAGMGGVGVM QDLAYAIPGI DEAMSFAEVL KQVNSLSYET IVFDTAPTGH TLRFLQFPTV L EKALAKVS QLSGQYGSLL NGILGGSGTL PNGQTLSDVM EKLDSLRVTI SEVNAQFKDE RLTTFVCVCI PEFLSLYETE RM IQELANY GIDTHCIVVN QLLFPKPGSD CEQCTARRRM QKKYLDQIEE LYDEEFNVVK MPLLVEEVRG KERLEKFSEM LIK PFVPPE WSHPQFEK

UniProtKB: Arsenite translocating ATPase-like protein

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Macromolecule #2: Protein GET2,Protein GET1

MacromoleculeName: Protein GET2,Protein GET1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 45.573727 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGRPTPLWRF LHTLLAVALG LAVIMLSPFG GTKLERDRAA AAVAGSASER EWLASLTDSY PLVKTGLGGG LFWAFATGEA ILLGTRWLF LSKKKKAATA AAKVNNNNGE GDDAELDSVE QAIELALEFF PAIRQPVEYL RPKVAVAMRY VDVGMTLWRD V MLALFVLG ...String:
MGRPTPLWRF LHTLLAVALG LAVIMLSPFG GTKLERDRAA AAVAGSASER EWLASLTDSY PLVKTGLGGG LFWAFATGEA ILLGTRWLF LSKKKKAATA AAKVNNNNGE GDDAELDSVE QAIELALEFF PAIRQPVEYL RPKVAVAMRY VDVGMTLWRD V MLALFVLG AVAWWRAGSG SENLYFQSGS GSMSLLLVIF LLELVVQLVN TIGAKTINNL LWRFYLSIPG SPLAKDFAEQ RA KQKEYLQ VRHDLNATSS QDEFAKWARL QRKHDKLMDE LEKKKSQLDA HRTSFSRKLT IYRWILTRGM QWFLCFWFSS QPM FWLPYG WFPYWVEWLV SFPNAPMGSV SIVVWQSACS GVLALVIEAV MAVVRYTGGT GMQKQRQPVP AAGGAPGTSK KDLG SGSLE VLFQ

UniProtKB: Uncharacterized protein, Protein GET1

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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

Concentration2.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
150.0 mMSodium Chloride
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 a Msp1E3D1 bound nanodisc

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 11997 / Average exposure time: 2.6 sec. / Average electron dose: 60.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5218800
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model generated in cryoSPARC
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)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 259692
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8odv:
Chaetomium thermophilum Get1/Get2 heterotetramer in complex with a Get3 dimer (nanodisc)

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