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- EMDB-25374: Get3 bound to ADP and the transmembrane domain of the tail-anchor... -

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Basic information

Entry
Database: EMDB / ID: EMD-25374
TitleGet3 bound to ADP and the transmembrane domain of the tail-anchored protein Bos1 - overall map
Map data
Sample
  • Complex: The targeting factor Get3 bound to the TMD of the cargo tail-anchored protein Bos1
    • Complex: The TMD of the tail-anchored protein Bos1
      • Protein or peptide: The TMD of the tail-anchored protein Bos1
    • Complex: The targeting factor Get3
      • Protein or peptide: ATPase GET3
KeywordsTail-anchored membrane protein targeting Deviant Walker A ATPase targeting factor / CHAPERONE
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsFry MY / Maggiolo AO / Clemons Jr WM
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-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25374.png

Downloads & links

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Map

FileDownload / File: emd_25374.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: 0.00427
Minimum - Maximum-0.009473245 - 0.019710349
Average (Standard dev.)-0.0000059233607 (±0.00053698703)
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_25374_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_25374_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_25374_half_map_2.map
Projections & Slices
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Sample components

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Entire : The targeting factor Get3 bound to the TMD of the cargo tail-anch...

EntireName: The targeting factor Get3 bound to the TMD of the cargo tail-anchored protein Bos1
Components
  • Complex: The targeting factor Get3 bound to the TMD of the cargo tail-anchored protein Bos1
    • Complex: The TMD of the tail-anchored protein Bos1
      • Protein or peptide: The TMD of the tail-anchored protein Bos1
    • Complex: The targeting factor Get3
      • Protein or peptide: ATPase GET3

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Supramolecule #1: The targeting factor Get3 bound to the TMD of the cargo tail-anch...

SupramoleculeName: The targeting factor Get3 bound to the TMD of the cargo tail-anchored protein Bos1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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 GET3

MacromoleculeName: ATPase GET3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLPSLHDILD QHTYKWIFFG GKGGVGKTTT SSSFSVLMAE TRPNEKFLLL STDPAHNISD AFDQKFGKAP TQVSGIPNLY AMEVDASNEM KSAVEAVQKE TGSAADNDAE SKSEGDMFGG LNDLITCASS FIKDGTFPGM DEMWSFINLI KLIDTNEYST VIFDTAPTGH ...String:
MLPSLHDILD QHTYKWIFFG GKGGVGKTTT SSSFSVLMAE TRPNEKFLLL STDPAHNISD AFDQKFGKAP TQVSGIPNLY AMEVDASNEM KSAVEAVQKE TGSAADNDAE SKSEGDMFGG LNDLITCASS FIKDGTFPGM DEMWSFINLI KLIDTNEYST VIFDTAPTGH TLRFLELPET VNKVLEIFTR LKDNMGGMLS MVMQTMGLSQ NDIFGLIDKT YPKIDVVKRI SAEFRDPSLC TFVGVCIPEF LSLYETERLV QRLAVLDMDC HAIVINFVLD ANAATPCSMC RSRARMQNKY IDQINELYDD FNIVLSPLRH DEVRGIANLR DYAETLIKPY RFCWSANPDP SSAK

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

MacromoleculeName: The TMD of the tail-anchored protein Bos1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
QKYLLVFWIA LILLIIGIYY VL

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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: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 73300
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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