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- PDB-7sq0: Get3 bound to ADP and the transmembrane domain of the tail-anchor... -

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Basic information

Entry
Database: PDB / ID: 7sq0
TitleGet3 bound to ADP and the transmembrane domain of the tail-anchored protein Bos1
Components
  • ATPase ASNA1 homolog
  • TMD of the tail-anchored protein Bos1
KeywordsCHAPERONE / Tail-anchored membrane protein targeting Deviant Walker A ATPase targeting factor
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
ADENOSINE-5'-DIPHOSPHATE / ATPase ASNA1 homolog
Similarity search - Component
Biological speciesGiardia intestinalis (eukaryote)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFry, M.Y. / Maggiolo, A.O. / Clemons Jr., W.M.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATPase ASNA1 homolog
B: ATPase ASNA1 homolog
C: TMD of the tail-anchored protein Bos1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,1588
Polymers94,1903
Non-polymers9685
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ATPase ASNA1 homolog / Arsenical pump-driving ATPase homolog / Arsenite-stimulated ATPase


Mass: 39164.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)
Strain: ATCC 50803 / WB clone C6 / Gene: GL50803_7953 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Nico21(DE3)
References: UniProt: A8B3G9, Hydrolases; Acting on acid anhydrides
#2: Protein TMD of the tail-anchored protein Bos1


Mass: 15860.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Nico21(DE3)
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1The targeting factor Get3 bound to ADP and the TMD of the cargo tail-anchored protein Bos1COMPLEX#1-#20MULTIPLE SOURCES
2The TMD of the tail-anchored protein Bos1COMPLEX#21RECOMBINANT
3The targeting factor Get3COMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.157 MDaYES
330.078 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Saccharomyces cerevisiae (brewer's yeast)4932
23Giardia intestinalis (strain ATCC 50803 / WB clone C6) (eukaryote)184922ATCC 50803 / WB clone C6
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Escherichia coli BL21(DE3) (bacteria)469008Nico21(DE3)
23Escherichia coli BL21(DE3) (bacteria)469008Nico21(DE3)
Buffer solutionpH: 7.5
SpecimenConc.: 0.73 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: DARK FIELD / Nominal magnification: 105000 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.82 sec. / Electron dose: 63.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2732
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.2.0particle selection
4CTFFIND4.1CTF correction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1790962
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70330 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 84.72 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029258
ELECTRON MICROSCOPYf_angle_d1.0616509
ELECTRON MICROSCOPYf_dihedral_angle_d14.4143860
ELECTRON MICROSCOPYf_chiral_restr0.044786
ELECTRON MICROSCOPYf_plane_restr0.0011384

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