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- PDB-8u1h: Axle-less Bacillus sp. PS3 F1 ATPase mutant -

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Basic information

Entry
Database: PDB / ID: 8u1h
TitleAxle-less Bacillus sp. PS3 F1 ATPase mutant
Components
  • (ATP synthase subunit ...) x 2
  • ATP synthase gamma chain
KeywordsHYDROLASE / ATPase / ATP synthase
Function / homology
Function and homology information


proton motive force-driven plasma membrane ATP synthesis / H+-transporting two-sector ATPase / proton-transporting ATP synthase complex / proton-transporting ATP synthase activity, rotational mechanism / ADP binding / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
ATP synthase, F1 complex, gamma subunit conserved site / ATP synthase gamma subunit signature. / ATP synthase, F1 complex, beta subunit / ATP synthase, alpha subunit, C-terminal domain superfamily / : / ATP synthase, F1 complex, gamma subunit / ATP synthase, F1 complex, gamma subunit superfamily / ATP synthase / ATP synthase, F1 complex, alpha subunit nucleotide-binding domain / ATP synthase, alpha subunit, C-terminal ...ATP synthase, F1 complex, gamma subunit conserved site / ATP synthase gamma subunit signature. / ATP synthase, F1 complex, beta subunit / ATP synthase, alpha subunit, C-terminal domain superfamily / : / ATP synthase, F1 complex, gamma subunit / ATP synthase, F1 complex, gamma subunit superfamily / ATP synthase / ATP synthase, F1 complex, alpha subunit nucleotide-binding domain / ATP synthase, alpha subunit, C-terminal / ATP synthase, F1 complex, alpha subunit / ATP synthase alpha/beta chain, C terminal domain / : / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / C-terminal domain of V and A type ATP synthase / ATP synthase subunit alpha, N-terminal domain-like superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain / ATP synthase alpha/beta family, beta-barrel domain / ATPase, alpha/beta subunit, nucleotide-binding domain, active site / ATP synthase alpha and beta subunits signature. / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / PHOSPHATE ION / ATP synthase subunit alpha / ATP synthase gamma chain / ATP synthase subunit beta
Similarity search - Component
Biological speciesBacillus sp. PS3 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsFurlong, E.J. / Zeng, Y.C. / Brown, S.H.J. / Sobti, M. / Stewart, A.G.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)2016308 Australia
CitationJournal: Biochim Biophys Acta Bioenerg / Year: 2025
Title: The molecular structure of an axle-less F-ATPase.
Authors: Emily J Furlong / Ian-Blaine P Reininger-Chatzigiannakis / Yi C Zeng / Simon H J Brown / Meghna Sobti / Alastair G Stewart /
Abstract: FF ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F-ATPase catalytic cores can hydrolyse ATP, passing through a series of ...FF ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic β subunits. Cooperativity in F-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and β subunits being identified. Single molecule studies have demonstrated that the F complexes lacking the γ axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open β subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F-ATPase.
History
DepositionSep 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Nov 20, 2024Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP synthase subunit alpha
B: ATP synthase subunit alpha
C: ATP synthase subunit alpha
D: ATP synthase subunit beta
E: ATP synthase subunit beta
F: ATP synthase subunit beta
G: ATP synthase gamma chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)357,41618
Polymers354,6777
Non-polymers2,73911
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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ATP synthase subunit ... , 2 types, 6 molecules ABCDEF

#1: Protein ATP synthase subunit alpha


Mass: 54908.676 Da / Num. of mol.: 3 / Mutation: C193S, W463F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. PS3 (bacteria) / Gene: uncA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0M3VGF9
#2: Protein ATP synthase subunit beta


Mass: 53424.625 Da / Num. of mol.: 3 / Mutation: Addition of His10 tag on N-term
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. PS3 (bacteria) / Gene: uncD / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0M4U1P9

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Protein , 1 types, 1 molecules G

#3: Protein ATP synthase gamma chain


Mass: 29677.084 Da / Num. of mol.: 1 / Mutation: S109C, I212C
Source method: isolated from a genetically manipulated source
Details: 4 residues deleted from N-term, 25 residues deleted from C-term, S109C, I212C, Strep-tag II insertion at N197
Source: (gene. exp.) Bacillus sp. PS3 (bacteria) / Gene: uncG / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0M4TPJ7

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Non-polymers , 4 types, 11 molecules

#4: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4

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Details

Has protein modificationN

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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

ComponentName: Bacillus PS3 F1 ATPase complex, with truncated gamma subunit
Type: COMPLEX
Details: The first 4 and last 25 amino acids have been deleted from the gamma subunit
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.354 MDa / Experimental value: NO
Source (natural)Organism: Bacillus sp. PS3 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Details: 100 mM Potassium phosphate pH 7, 2 mM EDTA 1 mM AMPPNP was added 20 min prior to freezing
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid was glow discharged using a PELCO easiGlow set to 15 mA
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10ISOLDEmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARC4.0.1classification
14cryoSPARC4.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23813 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: ModelAngelo was used to build an initial model into the map
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 65.25 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003723090
ELECTRON MICROSCOPYf_angle_d0.595531293
ELECTRON MICROSCOPYf_chiral_restr0.04473611
ELECTRON MICROSCOPYf_plane_restr0.00454076
ELECTRON MICROSCOPYf_dihedral_angle_d7.76483236

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