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- EMDB-25375: Nucleotide-free Get3 in the closed form -

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Basic information

Entry
Database: EMDB / ID: EMD-25375
TitleNucleotide-free Get3 in the closed form
Map data
Sample
  • Complex: Nucleotide-free Get3 in a closed conformation
    • Protein or peptide: ATPase GET3
KeywordsTail-anchored membrane protein targeting Deviant Walker A ATPase targeting factor / CHAPERONE
Biological speciesGiardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.46 Å
AuthorsFry MY / 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_25375.png

Downloads & links

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Map

FileDownload / File: emd_25375.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 192 pix.
= 203.328 Å		1.06 Å/pix.
x 192 pix.
= 203.328 Å		1.06 Å/pix.
x 192 pix.
= 203.328 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00845
Minimum - Maximum-0.028857885 - 0.043662414
Average (Standard dev.)0.000012045649 (±0.0014394416)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 203.328 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25375_msk_1.map
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Half map: #1

Fileemd_25375_half_map_1.map
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Half map: #2

Fileemd_25375_half_map_2.map
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Sample components

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Entire : Nucleotide-free Get3 in a closed conformation

EntireName: Nucleotide-free Get3 in a closed conformation
Components
  • Complex: Nucleotide-free Get3 in a closed conformation
    • Protein or peptide: ATPase GET3

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Supramolecule #1: Nucleotide-free Get3 in a closed conformation

SupramoleculeName: Nucleotide-free Get3 in a closed conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.55 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 Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 9300 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DARK FIELD / Nominal magnification: 88000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 11596225
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.46 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51340
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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