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Yorodumi- PDB-8dzf: Cryo-EM structure of bundle-forming pilus extension ATPase from E... -
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-Basic information
Entry | Database: PDB / ID: 8dzf | ||||||||||||
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Title | Cryo-EM structure of bundle-forming pilus extension ATPase from E.coli in the presence of AMP-PNP (class-2) | ||||||||||||
Components | BfpD | ||||||||||||
Keywords | STRUCTURAL PROTEIN / BfpD / AAA-ATPase / Type 4 pilus accessory protein | ||||||||||||
Function / homology | Type II/IV secretion system protein / Type II/IV secretion system protein / ATP hydrolysis activity / P-loop containing nucleoside triphosphate hydrolase / ATP binding / plasma membrane / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / BfpD Function and homology information | ||||||||||||
Biological species | Escherichia coli (E. coli) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.69 Å | ||||||||||||
Authors | Nayak, A.R. / Zhao, J. / Donnenberg, M.S. / Samso, M. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8dzf.cif.gz | 433.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8dzf.ent.gz | 354 KB | Display | PDB format |
PDBx/mmJSON format | 8dzf.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/dz/8dzf ftp://data.pdbj.org/pub/pdb/validation_reports/dz/8dzf | HTTPS FTP |
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-Related structure data
Related structure data | 27796MC 8dzeC 8dzgC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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-ZN / #3: Chemical | ChemComp-ANP / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Bundle-forming pilus extension ATPase from enteropathogenic E.coli in the presence of AMP-PNP Type: COMPLEX Details: BfpD ATPase with 5 mM Mgcl2, 1 mM ADP, 2 mM ATP and 2.5 mM AMP-PNP Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.362 MDa / Experimental value: YES |
Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.6 Details: 20 mM Tris (pH 7.6), 0.1 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.5 mM CHAPSO, 1 mM ADP, 2 mM ATP and 2.5 mM AMP-PNP |
Specimen | Conc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 10132 |
EM imaging optics | Energyfilter slit width: 10 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2831199 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68944 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL |