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- PDB-7spy: Get3 bound to ATP from G. intestinalis in the closed form -

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Basic information

Entry
Database: PDB / ID: 7spy
TitleGet3 bound to ATP from G. intestinalis in the closed form
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
ADENOSINE-5'-TRIPHOSPHATE / ATPase ASNA1 homolog
Similarity search - Component
Biological speciesGiardia intestinalis (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.23 Å
AuthorsFry, M.Y. / Maggiolo, A.O. / Clemons Jr., W.M.
Funding support1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM097572
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,6386
Polymers39,8791
Non-polymers7585
Water3,207178
1
A: ATPase ASNA1 homolog
hetero molecules

A: ATPase ASNA1 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,27512
Polymers79,7592
Non-polymers1,51710
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_555x-y,-y,-z+1/31
Buried area5670 Å2
ΔGint-120 kcal/mol
Surface area26980 Å2
MethodPISA
Unit cell
Length a, b, c (Å)79.646, 79.646, 130.070
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221
Components on special symmetry positions
IDModelComponents
11A-403-

ZN

21A-404-

ZN

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Components

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Protein , 1 types, 1 molecules A

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


Mass: 39879.340 Da / Num. of mol.: 1 / Mutation: D53N
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

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Non-polymers , 5 types, 183 molecules

#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 178 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.99 Å3/Da / Density % sol: 58.81 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1 M Tris, pH 7.5, 0.2 M ammonium sulfate, 15% 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: Jul 15, 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: 2.23→50 Å / Num. obs: 23301 / % possible obs: 97.4 % / Redundancy: 8.9 % / Rpim(I) all: 0.035 / Net I/σ(I): 22.56
Reflection shellResolution: 2.23→2.27 Å / Num. unique obs: 1163 / Rpim(I) all: 0.453

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
PHENIXv1.16refinement
HKL-3000data reduction
PHASERphasing
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3ibg

3ibg


Resolution: 2.23→43.39 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.939 / SU B: 5.298 / SU ML: 0.127 / Cross valid method: THROUGHOUT / ESU R: 0.203 / ESU R Free: 0.169 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.20886 1199 5.1 %RANDOM
Rwork0.17763 ---
obs0.17929 22102 97.42 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 37.73 Å2
Baniso -1Baniso -2Baniso -3
1--0.04 Å2-0.02 Å20 Å2
2---0.04 Å2-0 Å2
3---0.14 Å2
Refinement stepCycle: 1 / Resolution: 2.23→43.39 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2539 0 39 178 2756
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0132636
X-RAY DIFFRACTIONr_bond_other_d0.0010.0152462
X-RAY DIFFRACTIONr_angle_refined_deg1.3131.6433579
X-RAY DIFFRACTIONr_angle_other_deg1.2241.5785676
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2335322
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.00922.748131
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.28715450
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.8931514
X-RAY DIFFRACTIONr_chiral_restr0.0590.2356
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.022936
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02601
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_it2.3693.871288
X-RAY DIFFRACTIONr_mcbond_other2.3693.8681287
X-RAY DIFFRACTIONr_mcangle_it3.9055.7871607
X-RAY DIFFRACTIONr_mcangle_other3.9045.791608
X-RAY DIFFRACTIONr_scbond_it2.774.1311347
X-RAY DIFFRACTIONr_scbond_other2.774.1241343
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.536.0431966
X-RAY DIFFRACTIONr_long_range_B_refined6.88946.4183008
X-RAY DIFFRACTIONr_long_range_B_other6.80346.2212959
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.23→2.287 Å
RfactorNum. reflection% reflection
Rfree0.322 54 -
Rwork0.297 1265 -
obs--75.8 %

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