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- PDB-8dzg: Cryo-EM structure of bundle-forming pilus extension ATPase from E... -

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Basic information

Entry
Database: PDB / ID: 8dzg
TitleCryo-EM structure of bundle-forming pilus extension ATPase from E.coli in the presence of ADP
ComponentsBfpD
KeywordsSTRUCTURAL PROTEIN / BfpD / AAA-ATPase / Type 4 pilus accessory protein
Function / homologyType II/IV secretion system protein / Type II/IV secretion system protein / ATP hydrolysis activity / P-loop containing nucleoside triphosphate hydrolase / ATP binding / plasma membrane / ADENOSINE-5'-DIPHOSPHATE / BfpD
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsNayak, A.R. / Zhao, J. / Donnenberg, M.S. / Samso, M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)R01 AR068431 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116790 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R56A/111767 United States
CitationJournal: mBio / Year: 2022
Title: Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.
Authors: Ashok R Nayak / Pradip K Singh / Jinlei Zhao / Montserrat Samsó / Michael S Donnenberg /
Abstract: Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ...Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.
History
DepositionAug 7, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 4, 2023Group: 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 1.3Jan 11, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: BfpD
B: BfpD
C: BfpD
D: BfpD
E: BfpD
F: BfpD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)365,74224
Polymers362,6406
Non-polymers3,10118
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Negative staining
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
BfpD


Mass: 60440.062 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bfpD / Production host: Escherichia coli (E. coli) / References: UniProt: Q47070
#2: Chemical
ChemComp-MG / MAGNESIUM ION


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


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestY

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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: Bundle-forming pilus extension ATPase from enteropathogenic E. coli in the presence of ADP
Type: ORGANELLE OR CELLULAR COMPONENT / Details: BfpD ATPase with 5 mM MgCl2 and 1 mM ADP / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.362 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Details: 20 mM Tris (pH 7.6), 0.1 mM NaCl, 1 mM ADP, 5 mM MgCl2, 2 mM DTT, 0.5 mM CHAPSO
SpecimenConc.: 4 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 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 8387
EM imaging opticsEnergyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARC3.2CTF correction
7Coot0.9.5.8model fitting
9PHENIX1.20rc4-4425model refinement
13cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1135477
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 317950 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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