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- PDB-8zi3: Cryo-EM reveals transition states of the Acinetobacter baumannii ... -

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Basic information

Entry
Database: PDB / ID: 8zi3
TitleCryo-EM reveals transition states of the Acinetobacter baumannii F1-ATPase rotary subunits gamma and epsilon and novel compound targets - Conformation 4
Components(ATP synthase ...) x 4
KeywordsHYDROLASE / ATP hydrolysis / F1-ATPase
Function / homology
Function and homology information


proton motive force-driven plasma membrane ATP synthesis / proton-transporting ATP synthase complex, catalytic core F(1) / H+-transporting two-sector ATPase / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / hydrolase activity / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
ATP synthase delta/epsilon subunit, C-terminal domain superfamily / ATP synthase, F1 complex, delta/epsilon subunit / ATP synthase, F1 complex, delta/epsilon subunit, N-terminal / F0F1 ATP synthase delta/epsilon subunit, N-terminal / ATP synthase, Delta/Epsilon chain, beta-sandwich domain / 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 delta/epsilon subunit, C-terminal domain superfamily / ATP synthase, F1 complex, delta/epsilon subunit / ATP synthase, F1 complex, delta/epsilon subunit, N-terminal / F0F1 ATP synthase delta/epsilon subunit, N-terminal / ATP synthase, Delta/Epsilon chain, beta-sandwich domain / 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, alpha subunit, C-terminal / ATP synthase, F1 complex, alpha subunit / ATP synthase, F1 complex, alpha subunit nucleotide-binding domain / 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 / ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / ATP synthase subunit alpha / ATP synthase gamma chain / ATP synthase epsilon chain / ATP synthase subunit beta
Similarity search - Component
Biological speciesAcinetobacter baumannii AB5075 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsLe, K.C.M. / Wong, C.F. / Gruber, G.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)RG20/22 Singapore
CitationJournal: FASEB J / Year: 2024
Title: Cryo-EM reveals transition states of the Acinetobacter baumannii F-ATPase rotary subunits γ and ε, unveiling novel compound targets.
Authors: Khoa Cong Minh Le / Chui Fann Wong / Volker Müller / Gerhard Grüber /
Abstract: Priority 1: critical WHO pathogen Acinetobacter baumannii depends on ATP synthesis and ATP:ADP homeostasis and its bifunctional FF-ATP synthase. While synthesizing ATP, it regulates ATP cleavage by ...Priority 1: critical WHO pathogen Acinetobacter baumannii depends on ATP synthesis and ATP:ADP homeostasis and its bifunctional FF-ATP synthase. While synthesizing ATP, it regulates ATP cleavage by its inhibitory ε subunit to prevent wasteful ATP consumption. We determined cryo-electron microscopy structures of the ATPase active A. baumannii F-αßγε mutant in four distinct conformational states, revealing four transition states and structural transformation of the ε's C-terminal domain, forming the switch of an ATP hydrolysis off- and an ATP synthesis on-state based. These alterations go in concert with altered motions and interactions in the catalytic- and rotary subunits of this engine. These A. baumannii interacting sites provide novel pathogen-specific targets for inhibitors, with the aim of ATP depletion and/or ATP synthesis and growth inhibition. Furthermore, the presented diversity to other bacterial F-ATP synthases extends the view of structural elements regulating such a catalyst.
History
DepositionMay 12, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)365,90719
Polymers363,3148
Non-polymers2,59211
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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ATP synthase ... , 4 types, 8 molecules eBCADFEg

#1: Protein ATP synthase epsilon chain / ATP synthase F1 sector epsilon subunit / F-ATPase epsilon subunit


Mass: 13838.840 Da / Num. of mol.: 1 / Mutation: deletion 134-139
Source method: isolated from a genetically manipulated source
Details: Subunit epsilon with truncated C-terminus domain, deletion I134-Q139
Source: (gene. exp.) Acinetobacter baumannii AB5075 (bacteria)
Gene: atpC, A0141_RS12850, A0141_RS20965, A7M90_08520, Aba7835_18745, ABCAM1_0172, ABR2091_0173, ABUW_3731, ACX61_17950, APD17_18795, AUO97_06420, AYR68_18050, B7L45_18620, B9W25_04440, B9X95_01230, ...Gene: atpC, A0141_RS12850, A0141_RS20965, A7M90_08520, Aba7835_18745, ABCAM1_0172, ABR2091_0173, ABUW_3731, ACX61_17950, APD17_18795, AUO97_06420, AYR68_18050, B7L45_18620, B9W25_04440, B9X95_01230, C2U32_15275, CAS83_08020, CBE85_14435, CBL15_17785, CSB70_3895, CTZ19_18500, CV954_019155, D8O08_000335, DLI71_10775, DOL94_02920, E1A86_02075, E1A87_05110, EA686_08570, EA706_05510, EA720_009765, EA722_10625, EGM95_19705, F2P40_12650, F4T85_15175, FDN00_02385, FE003_18665, FJU42_13255, FJV09_08795, FR761_02125, G3N53_14500, GNY86_14290, GSE42_00725, HB367_12610, HBK86_18985, HIN86_18905, IMO23_00750, JHZ39_002241, P9867_05900, SAMEA104305318_03328, SAMEA4394745_01411
Plasmid: pET29b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: V5VHG0
#2: Protein ATP synthase subunit alpha / ATP synthase F1 sector subunit alpha / F-ATPase subunit alpha


Mass: 55452.906 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii AB5075 (bacteria)
Gene: atpA, A1S_0153 / Plasmid: pET29b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: A3M142, H+-transporting two-sector ATPase
#3: Protein ATP synthase subunit beta / ATP synthase F1 sector subunit beta / F-ATPase subunit beta


Mass: 50327.180 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii AB5075 (bacteria)
Gene: atpD, A0141_RS12845, A0141_RS20960, A7M90_08515, Aba7835_18750, ABCAM1_0171, ABR2091_0172, ABUW_3732, ACX61_17955, APD17_18800, AUO97_06425, AYR68_18055, B7L45_18625, B9W25_04445, B9X95_01225, ...Gene: atpD, A0141_RS12845, A0141_RS20960, A7M90_08515, Aba7835_18750, ABCAM1_0171, ABR2091_0172, ABUW_3732, ACX61_17955, APD17_18800, AUO97_06425, AYR68_18055, B7L45_18625, B9W25_04445, B9X95_01225, C2U32_15270, CAS83_08025, CBE85_14440, CBL15_17790, CSB70_3894, CTZ19_18505, CV954_019160, D8O08_000340, DLI71_10780, DOL94_02915, E1A86_02070, E1A87_05115, EA706_05505, EA720_009770, EA722_10620, EGM95_19710, F2P40_12655, F4T85_15180, FDN00_02380, FE003_18670, FJU42_13260, FJV09_08800, FR761_02120, G3N53_14495, GNY86_14295, GSE42_00720, HB367_12615, HBK86_18980, HIN86_18910, IAG11_01885, IMO23_00745, ITE13_08765, JHZ39_002240, P9867_05895, SAMEA104305318_03329, SAMEA4394745_01410
Plasmid: pET29b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: V5VHQ6, H+-transporting two-sector ATPase
#4: Protein ATP synthase gamma chain / ATP synthase F1 sector gamma subunit / F-ATPase gamma subunit


Mass: 32135.037 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii AB5075 (bacteria)
Gene: atpG, A1S_0154 / Plasmid: pET29b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: A3M143

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Non-polymers , 4 types, 11 molecules

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


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


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#8: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: Acinetobacter baumannii F1-ATPase recombinant protein with truncation mutation at subunit epsilon CTD, 134-139
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.37 MDa / Experimental value: NO
Source (natural)Organism: Acinetobacter baumannii AB5075 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41
Buffer solutionpH: 7.5 / Details: 50 mM Tris-HCl, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2150 mMNaClNaCl1
SpecimenConc.: 1.5 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: 277.15 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 160000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.02 sec. / Electron dose: 68.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8635

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selection
2SerialEMimage acquisition
4cryoSPARC4.2.1CTF correction
7UCSF ChimeraX1.6.1model fittingUCSF ChimeraX was used to rough fit the model into the map
8Coot0.9.8.1model fittingCoot was used to rigid body fit all residues individually
10Coot0.9.8.1model refinementCoot was used to real-space refine all residues individually
11PHENIX1.20.1-4487model refinementPhenix was used to real-space refine the entire model in the map
12cryoSPARC4.2.1initial Euler assignment
13cryoSPARC4.2.1final Euler assignment
14cryoSPARC4.2.1classification
15cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 829861
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138690 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 7YRY

7yry


Accession code: 7YRY
Details: The initial model is from the CryoEM structure of AbF1-ATPase Wild Type form.
Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00225052
ELECTRON MICROSCOPYf_angle_d0.53133963
ELECTRON MICROSCOPYf_dihedral_angle_d4.7563541
ELECTRON MICROSCOPYf_chiral_restr0.0423916
ELECTRON MICROSCOPYf_plane_restr0.0034418

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