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- PDB-7spz: Nucleotide-free Get3 in two open forms -

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Basic information

Entry
Database: PDB / ID: 7spz
TitleNucleotide-free Get3 in two open forms
ComponentsATPase ASNA1 homolog
KeywordsCHAPERONE / Tail-anchored membrane protein targeting Deviant Walker A ATPase targeting factor
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides / protein insertion into ER membrane / endoplasmic reticulum / 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 speciesGiardia lamblia ATCC 50803 (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 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 1.3Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,5244
Polymers78,3932
Non-polymers1312
Water00
1
A: ATPase ASNA1 homolog
hetero molecules

A: ATPase ASNA1 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,5244
Polymers78,3932
Non-polymers1312
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,-y,z1
Buried area2550 Å2
ΔGint-78 kcal/mol
Surface area31010 Å2
MethodPISA
2
B: ATPase ASNA1 homolog
hetero molecules

B: ATPase ASNA1 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,5244
Polymers78,3932
Non-polymers1312
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-x,-y+1,z1
Buried area3050 Å2
ΔGint-77 kcal/mol
Surface area28370 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.138, 102.605, 138.801
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-400-

ZN

21B-400-

ZN

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Components

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


Mass: 39196.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Giardia lamblia ATCC 50803 (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: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.42 Å3/Da / Density % sol: 49.1 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 5.3
Details: 0.1 M MES, pH 5.3, 0.1 M magnesium chloride, 21% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 19, 2021
RadiationMonochromator: Liquid nitrogen-cooled double crystal Si(111)
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2.77→50 Å / Num. obs: 17087 / % possible obs: 84.2 % / Redundancy: 11.5 % / CC1/2: 0.994 / Net I/σ(I): 17.9
Reflection shellResolution: 2.77→2.82 Å / Num. unique obs: 997 / CC1/2: 0.511

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
HKL-3000data reduction
SCALEPACKdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3IBG

3ibg


Resolution: 3→28.11 Å / Cor.coef. Fo:Fc: 0.831 / Cor.coef. Fo:Fc free: 0.768 / SU B: 26.846 / SU ML: 0.492 / Cross valid method: THROUGHOUT / ESU R Free: 0.659 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.3483 709 4.8 %RANDOM
Rwork0.29314 ---
obs0.2958 14025 91.16 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 44.526 Å2
Baniso -1Baniso -2Baniso -3
1-1.68 Å2-0 Å20 Å2
2---0.21 Å2-0 Å2
3----1.46 Å2
Refinement stepCycle: 1 / Resolution: 3→28.11 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5072 0 2 0 5074
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0020.0135177
X-RAY DIFFRACTIONr_bond_other_d0.0010.0154890
X-RAY DIFFRACTIONr_angle_refined_deg1.1521.647006
X-RAY DIFFRACTIONr_angle_other_deg1.0151.57811276
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.2445635
X-RAY DIFFRACTIONr_dihedral_angle_2_deg29.82722.814263
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.33215902
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.7071528
X-RAY DIFFRACTIONr_chiral_restr0.0320.2695
X-RAY DIFFRACTIONr_gen_planes_refined0.0020.025793
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021179
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.9554.7762558
X-RAY DIFFRACTIONr_mcbond_other0.9544.7752557
X-RAY DIFFRACTIONr_mcangle_it1.7827.1513187
X-RAY DIFFRACTIONr_mcangle_other1.7827.1533188
X-RAY DIFFRACTIONr_scbond_it0.5354.8352618
X-RAY DIFFRACTIONr_scbond_other0.5354.8352618
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other1.0697.2253820
X-RAY DIFFRACTIONr_long_range_B_refined3.53355.3675515
X-RAY DIFFRACTIONr_long_range_B_other3.53355.3395513
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 3→3.077 Å
RfactorNum. reflection% reflection
Rfree0.421 36 -
Rwork0.319 615 -
obs--56.22 %

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