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- EMDB-25373: Get3 bound to ADP and the transmembrane domain of the tail-anchor... -

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Basic information

Entry
Database: EMDB / ID: EMD-25373
TitleGet3 bound to ADP and the transmembrane domain of the tail-anchored protein Bos1 - nucleotide binding domain map
Map data
Sample
  • Complex: The targeting factor Get3 bound to ADP and the TMD of the cargo tail-anchored protein Bos1
    • Complex: The TMD of the tail-anchored protein Bos1
      • Protein or peptide: TMD of the tail-anchored protein Bos1
    • Complex: The targeting factor Get3
      • Protein or peptide: ATPase ASNA1 homolog
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsTail-anchored membrane protein targeting Deviant Walker A ATPase targeting factor / CHAPERONE
Function / homology
Function and homology information


GET complex / Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
Arsenical pump ATPase, ArsA/GET3, eukaryotic / Arsenical pump ATPase, ArsA/GET3 / Anion-transporting ATPase-like domain / Anion-transporting ATPase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATPase ASNA1 homolog
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFry MY / Maggiolo AO
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM097572 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structurally derived universal mechanism for the catalytic cycle of the tail-anchored targeting factor Get3.
Authors: Michelle Y Fry / Vladimíra Najdrová / Ailiena O Maggiolo / Shyam M Saladi / Pavel Doležal / William M Clemons /
Abstract: Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) ...Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. Here, we identify the GET pathway in Giardia intestinalis and present the structure of the Get3-client complex in the critical postnucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work resolves key conundrums and allows for a complete model of the dramatic conformational landscape of Get3.
History
DepositionNov 4, 2021-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25373.png

Downloads & links

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Map

FileDownload / File: emd_25373.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.866 Å
Density
Contour LevelBy AUTHOR: 0.0058
Minimum - Maximum-0.014666214 - 0.029405728
Average (Standard dev.)-0.0000047787616 (±0.00078824686)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 263.264 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25373_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: focused refined half map

Fileemd_25373_half_map_1.map
Annotationfocused refined half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: focused refined half map

Fileemd_25373_half_map_2.map
Annotationfocused refined half 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 : The targeting factor Get3 bound to ADP and the TMD of the cargo t...

EntireName: The targeting factor Get3 bound to ADP and the TMD of the cargo tail-anchored protein Bos1
Components
  • Complex: The targeting factor Get3 bound to ADP and the TMD of the cargo tail-anchored protein Bos1
    • Complex: The TMD of the tail-anchored protein Bos1
      • Protein or peptide: TMD of the tail-anchored protein Bos1
    • Complex: The targeting factor Get3
      • Protein or peptide: ATPase ASNA1 homolog
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: The targeting factor Get3 bound to ADP and the TMD of the cargo t...

SupramoleculeName: The targeting factor Get3 bound to ADP and the TMD of the cargo tail-anchored protein Bos1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 157 KDa

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Supramolecule #2: The TMD of the tail-anchored protein Bos1

SupramoleculeName: The TMD of the tail-anchored protein Bos1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: The targeting factor Get3

SupramoleculeName: The targeting factor Get3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Strain: ATCC 50803 / WB clone C6
Molecular weightTheoretical: 78 KDa

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Macromolecule #1: ATPase ASNA1 homolog

MacromoleculeName: ATPase ASNA1 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on acid anhydrides
Source (natural)Organism: Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Strain: ATCC 50803 / WB clone C6
Molecular weightTheoretical: 39.164922 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLPSLHDILD QHTYKWIFFG GKGGVGKTTT SSSFSVLMAE TRPNEKFLLL STDPAHNISD AFDQKFGKAP TQVSGIPNLY AMEVDASNE MKSAVEAVQK ETGSAA(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK) ...String:
MLPSLHDILD QHTYKWIFFG GKGGVGKTTT SSSFSVLMAE TRPNEKFLLL STDPAHNISD AFDQKFGKAP TQVSGIPNLY AMEVDASNE MKSAVEAVQK ETGSAA(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)ITCA SSFIKDGTFP GMDEMWSFIN LIKLIDTNEY STVIFD TAP TGHTLRFLEL PETVNKVLEI FTRLKDNMGG MLSMVMQTMG LSQNDIFGLI DKTYPKIDVV KRISAEFRDP SLCTFVG VC IPEFLSLYET ERLVQRLAVL DMDCHAIVIN FVLDANAATP CSMCRSRARM QNKYIDQINE LYDDFNIVLS PLRHDEVR G IANLRDYAET LIKPYRFCWS ANPDPSSAK

UniProtKB: ATPase ASNA1 homolog, ATPase ASNA1 homolog

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Macromolecule #2: TMD of the tail-anchored protein Bos1

MacromoleculeName: TMD of the tail-anchored protein Bos1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 15.860031 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MDHHHHHHEN LYFQSADLED NWETLNDNLK VIEKADNAAQ VKDALTKMRA AALDAQKATP PKLEDKSPDS PEMKDFRHGF DILVGQIDD ALKLANEGKV KEAQAAAEQL KTTRNAYIQK YLLVFWIALI LLIIGIYYVL

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration0.73 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 2732 / Average exposure time: 1.82 sec. / Average electron dose: 63.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DARK FIELD / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1790962
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70330
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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