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- EMDB-27796: Cryo-EM structure of bundle-forming pilus extension ATPase from E... -

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Basic information

Entry
Database: EMDB / ID: EMD-27796
TitleCryo-EM structure of bundle-forming pilus extension ATPase from E.coli in the presence of AMP-PNP (class-2)
Map data
Sample
  • Complex: Bundle-forming pilus extension ATPase from enteropathogenic E.coli in the presence of AMP-PNP
    • Protein or peptide: BfpD
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Function / homologyType II/IV secretion system protein / Type II/IV secretion system protein / P-loop containing nucleoside triphosphate hydrolase / ATP binding / BfpD
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsNayak AR / Zhao J / Donnenberg MS / Samso M
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27796.png

Downloads & links

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Map

FileDownload / File: emd_27796.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.3944417 - 0.693498
Average (Standard dev.)0.00166553 (±0.02585495)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27796_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27796_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Bundle-forming pilus extension ATPase from enteropathogenic E.col...

EntireName: Bundle-forming pilus extension ATPase from enteropathogenic E.coli in the presence of AMP-PNP
Components
  • Complex: Bundle-forming pilus extension ATPase from enteropathogenic E.coli in the presence of AMP-PNP
    • Protein or peptide: BfpD
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Bundle-forming pilus extension ATPase from enteropathogenic E.col...

SupramoleculeName: Bundle-forming pilus extension ATPase from enteropathogenic E.coli in the presence of AMP-PNP
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Details: BfpD ATPase with 5 mM Mgcl2, 1 mM ADP, 2 mM ATP and 2.5 mM AMP-PNP
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 362 KDa

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Macromolecule #1: BfpD

MacromoleculeName: BfpD / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 60.440062 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVNKTEKTSD LMFERFKRNV SEIVTGDGGE LELTVEQRKY FLIFKNGDFL VSSCHMKHHL VQMLREIATR KGYPNLTIYE VNLKDIRLL YEASLKTVQN NGQDLLPVEK RASMLLFECA EMRVSDLHIK VYDAEADIYI RKDGDMELLR QIESNTAHSI L ASLYNNAD ...String:
MVNKTEKTSD LMFERFKRNV SEIVTGDGGE LELTVEQRKY FLIFKNGDFL VSSCHMKHHL VQMLREIATR KGYPNLTIYE VNLKDIRLL YEASLKTVQN NGQDLLPVEK RASMLLFECA EMRVSDLHIK VYDAEADIYI RKDGDMELLR QIESNTAHSI L ASLYNNAD DSDATYKINA YQAARIVASK SRLALPPVIQ AVRLQFNPLG QGGRYLIARF LYTDKSEKQK EMDPTRFGFH HS HAESFSR MRNLPIGINI ISGPTGSGKS TTLKNLLELL YIEKKKKVNI ISIEDPPEYE IDGTAQLPIT NVETEAQRGE EYR KAITAA LRSDPDIIMP GEARDAEVIN LLFTAAMTGH QVWTSLHANN ALAIFDRLKD QGVDEFKLTD PELITGLVAQ RLVR KLCAQ CSITLTEYIA SGGGISDTDR KIISGHETSV RFPNPRAKKC CRDGYNGRTI LAEVIEPDSK LLRLVAEGKR EDAQH YWLT SLHGMALKEH AWLKIISGEI CVMDAVNKIS GIDNITEERK KYLFSRDNEI

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.5 mg/mL
BufferpH: 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
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Slit width: 10 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 3 / Number real images: 10132 / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2831199
Startup modelType of model: NONE / Details: Ab-initio reconstruction in cryosparc 2.15
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 2
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 68944
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8dzf:
Cryo-EM structure of bundle-forming pilus extension ATPase from E.coli in the presence of AMP-PNP (class-2)

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