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- PDB-7ul6: CryoEM structure of full-length dimeric ClbP -

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Basic information

Entry
Database: PDB / ID: 7ul6
TitleCryoEM structure of full-length dimeric ClbP
ComponentsBeta-lactamase
KeywordsHYDROLASE / colibactin peptidase / S12 peptidase / inner-membrane hydrolase
Function / homology
Function and homology information


antibiotic catabolic process / beta-lactamase activity / beta-lactamase / outer membrane-bounded periplasmic space / response to antibiotic / membrane
Similarity search - Function
Beta-lactamase, class-C active site / Beta-lactamase class-C active site. / : / Beta-lactamase-related / Beta-lactamase / Beta-lactamase / DD-peptidase/beta-lactamase superfamily / Beta-lactamase/transpeptidase-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesEscherichia coli CFT073 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsVelilla, J.A. / Walsh Jr., R.M. / Gaudet, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM120996 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA208834 United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: Structural basis of colibactin activation by the ClbP peptidase.
Authors: José A Velilla / Matthew R Volpe / Grace E Kenney / Richard M Walsh / Emily P Balskus / Rachelle Gaudet /
Abstract: Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the ...Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.
History
DepositionApr 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 2, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 15, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Oct 23, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-lactamase
B: Beta-lactamase


Theoretical massNumber of molelcules
Total (without water)107,0472
Polymers107,0472
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Solution structure suggests ClbP forms a dimer and no class evidences a monomer. Light scattering experiments also suggest the protein exists predominantly as a species ...Evidence: electron microscopy, Solution structure suggests ClbP forms a dimer and no class evidences a monomer. Light scattering experiments also suggest the protein exists predominantly as a species with an average molecular weight of 110 kDa.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Beta-lactamase / Colibactin peptidase


Mass: 53523.309 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli CFT073 (bacteria) / Plasmid: pET29b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41 / References: UniProt: Q0P7K6, beta-lactamase
Has protein modificationY

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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: ClbP / Type: COMPLEX / Details: Full-length ClbP / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.110 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli CFT073 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41 (DE3) / Plasmid: pET29b
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2200 mMsodium chlorideNaCl1
30.06 %GDN1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Protein was purified by Ni affinity chromatography followed by SEC on an S200 10/300 column equilibrated with 10 mM HEPES pH 7.3, 200 mM NaCl, 0.06% GDN. Sample used for preparing grids came ...Details: Protein was purified by Ni affinity chromatography followed by SEC on an S200 10/300 column equilibrated with 10 mM HEPES pH 7.3, 200 mM NaCl, 0.06% GDN. Sample used for preparing grids came from the peak fraction and was not concentrated.
Specimen supportDetails: 30 s glow discharge at 15mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: Three uL of sample were deposited onto 400 mesh Quantifoil Cu 1.2/1.3 grids that had been glow discharged in a PELCO easiGLOW (Ted Pella) at 0.39 mBar, 15 mA for 30 s. Samples were vitrified ...Details: Three uL of sample were deposited onto 400 mesh Quantifoil Cu 1.2/1.3 grids that had been glow discharged in a PELCO easiGLOW (Ted Pella) at 0.39 mBar, 15 mA for 30 s. Samples were vitrified in 100% liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific), with a wait time of 30 s, blot time of 5 s and a blot force of 16 at 100% humidity.

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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 FIELD / Nominal magnification: 105000 X / Calibrated magnification: 60606 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 76.191 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3888
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.8.6image acquisition
4RELION3.1.1CTF correction
7Coot0.93model fittingIterative manual fitting
8ISOLDE1.0b4.dev0model fittingIterative rebuilding
9UCSF Chimera1.15model fittingRigid body of starting model
12cryoSPARCfinal Euler assignment
14cryoSPARC3D reconstruction
15PHENIX1.20.1-4487model refinementReal space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 562462
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109906 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7MDF

7mdf


Pdb chain-ID: A / Accession code: 7MDF / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036796
ELECTRON MICROSCOPYf_angle_d0.5519264
ELECTRON MICROSCOPYf_dihedral_angle_d4.777944
ELECTRON MICROSCOPYf_chiral_restr0.0421080
ELECTRON MICROSCOPYf_plane_restr0.0061188

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