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- PDB-6njp: Structure of the assembled ATPase EscN in complex with its centra... -

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Basic information

Entry
Database: PDB / ID: 6njp
TitleStructure of the assembled ATPase EscN in complex with its central stalk EscO from the enteropathogenic E. coli (EPEC) type III secretion system
Components
  • EscO
  • Translocator EscN
KeywordsHYDROLASE / ATPase / Type III Secretion System / ADP / hexamer
Function / homology
Function and homology information


protein-secreting ATPase / type III protein secretion system complex / protein secretion by the type III secretion system / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / ATP hydrolysis activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Type 3 secretion system ATPase SCTN / ATPase, type III secretion system, FliI/YscN / T3SS EscN ATPase, C-terminal / T3SS EscN ATPase C-terminal 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 / ALUMINUM FLUORIDE / Uncharacterized protein / protein-secreting ATPase
Similarity search - Component
Biological speciesEscherichia coli O127:H6 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsMajewski, D.D. / Worrall, L.J. / Hong, C. / Atkinson, C.E. / Vuckovic, M. / Watanabe, N. / Yu, Z. / Strynadka, N.C.J.
Funding support Canada, United States, 3items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canadian Institutes of Health Research (CIHR) Canada
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2019
Title: Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry.
Authors: Dorothy D Majewski / Liam J Worrall / Chuan Hong / Claire E Atkinson / Marija Vuckovic / Nobuhiko Watanabe / Zhiheng Yu / Natalie C J Strynadka /
Abstract: Many Gram-negative bacteria, including causative agents of dysentery, plague, and typhoid fever, rely on a type III secretion system - a multi-membrane spanning syringe-like apparatus - for their ...Many Gram-negative bacteria, including causative agents of dysentery, plague, and typhoid fever, rely on a type III secretion system - a multi-membrane spanning syringe-like apparatus - for their pathogenicity. The cytosolic ATPase complex of this injectisome is proposed to play an important role in energizing secretion events and substrate recognition. We present the 3.3 Å resolution cryo-EM structure of the enteropathogenic Escherichia coli ATPase EscN in complex with its central stalk EscO. The structure shows an asymmetric pore with different functional states captured in its six catalytic sites, details directly supporting a rotary catalytic mechanism analogous to that of the heterohexameric F/V-ATPases despite its homohexameric nature. Situated at the C-terminal opening of the EscN pore is one molecule of EscO, with primary interaction mediated through an electrostatic interface. The EscN-EscO structure provides significant atomic insights into how the ATPase contributes to type III secretion, including torque generation and binding of chaperone/substrate complexes.
History
DepositionJan 3, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 20, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

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Assembly

Deposited unit
A: Translocator EscN
B: Translocator EscN
C: Translocator EscN
D: Translocator EscN
E: Translocator EscN
F: Translocator EscN
G: EscO
hetero molecules


Theoretical massNumber of molelcules
Total (without water)312,29219
Polymers310,1517
Non-polymers2,14212
Water37821
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29570 Å2
ΔGint-124 kcal/mol
Surface area91370 Å2
MethodPISA

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Components

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Protein , 2 types, 7 molecules ABCDEFG

#1: Protein
Translocator EscN


Mass: 49196.566 Da / Num. of mol.: 6 / Fragment: UNP residues 29-446
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria)
Strain: E2348/69 / EPEC / Gene: escN, E2348C_3948 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B7UMA6
#2: Protein EscO


Mass: 14971.122 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria)
Strain: E2348/69 / EPEC / Gene: E2348C_3947 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B7UMA5

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Non-polymers , 4 types, 33 molecules

#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


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


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-AF3 / ALUMINUM FLUORIDE / Aluminium fluoride


Mass: 83.977 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: AlF3
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Formula: H2O

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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: Homohexameric complex of ATPase EscN bound to one molecule of central stalk EscO
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli O127:H6 str. E2348/69 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.34 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 FIELDBright-field microscopy
Image recordingElectron dose: 57.67 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9cryoSPARC2initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 600000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: phenix.real_space_refine
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDPdb chain residue range
12OBM

2obm

2OBM1102-446
25B0O

5b0o

5B0O226-97

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