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- PDB-8xju: Cryo-EM structure of colibactin assembly line polyketide synthase... -

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Basic information

Entry
Database: PDB / ID: 8xju
TitleCryo-EM structure of colibactin assembly line polyketide synthase ClbI (apo state)
ComponentsPolyketide synthase
KeywordsTRANSFERASE / colibactin / microbiome / polyketide synthase / colorectal cancer / NRPS-PKS hybrid / biosynthetic protein
Function / homology
Function and homology information


beta-ketoacyl-[acyl-carrier-protein] synthase I / fatty acid synthase activity / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process
Similarity search - Function
Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Beta-ketoacyl synthase ...Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Beta-ketoacyl synthase / Ketosynthase family 3 (KS3) domain profile. / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / Thiolase-like / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsKim, M. / Kim, J. / Kang, J.Y.
Funding support Korea, Republic Of, 2items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF- 2019M3E5D6063908 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF- 2020R1F1A107746211 Korea, Republic Of
CitationJournal: Structure / Year: 2025
Title: Structural study on human microbiome-derived polyketide synthases that assemble genotoxic colibactin.
Authors: Minjae Kim / Jinwoo Kim / Gyu Sung Lee / Paul Dominic B Olinares / Yougant Airan / Jasmine L Chow / Jongseok Park / Yujin Jeong / Jiho Park / Brian T Chait / Seth B Herzon / Chung Sub Kim / Jin Young Kang /
Abstract: Colibactin, a human microbiome-derived genotoxin, promotes colorectal cancer by damaging the host gut epithelial genomes. While colibactin is synthesized via a hybrid non-ribosomal peptide synthetase ...Colibactin, a human microbiome-derived genotoxin, promotes colorectal cancer by damaging the host gut epithelial genomes. While colibactin is synthesized via a hybrid non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway, known as pks or clb, the structural details of its biosynthetic enzymes remain limited, hindering our understanding of its biosynthesis and clinical application. In this study, we report the cryo-EM structures of two colibactin-producing PKS enzymes, ClbC and ClbI, captured in different reaction states using a substrate-mimic crosslinker. Our structural analysis revealed the binding sites of carrier protein (CP) domains of the ClbC and ClbI on their ketosynthase (KS) domains. Further, we identified a novel NRPS-PKS docking interaction between ClbI and its upstream enzyme, ClbH, mediated by the C-terminal peptide ClbH and the dimeric interface of ClbI, establishing a 1:2 stoichiometry. These findings advance our understanding of colibactin assembly line and provide broader insights into NRPS-PKS natural product biosynthesis mechanisms.
History
DepositionDec 22, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.1May 28, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyketide synthase
B: Polyketide synthase


Theoretical massNumber of molelcules
Total (without water)221,3052
Polymers221,3052
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Polyketide synthase / Putative polyketide synthase


Mass: 110652.742 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: clbI, ppsE_5, EPS76_08200, EWK56_24760, FPI65_12365, HMW38_22985, IFB95_004343, NCTC9075_02731
Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q0P7J9, beta-ketoacyl-[acyl-carrier-protein] synthase I
Has protein modificationN

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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: apo-ClbI / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
220 mMTRIS-hydrochlorideTris-HCl1
310 mMmagnesium chlorideMgCl21
41 mMdithiothreitolDTT1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 58.85 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11100
Image scansWidth: 6000 / Height: 4000

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.0.1particle selection
2Leginonimage acquisition
4cryoSPARC3.0.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC3.0.1initial Euler assignment
11RELION3.1.1final Euler assignment
12cryoSPARC3.0.1classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2791165
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1037444 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0113240
ELECTRON MICROSCOPYf_angle_d1.12118040
ELECTRON MICROSCOPYf_dihedral_angle_d32.81834
ELECTRON MICROSCOPYf_chiral_restr0.0722063
ELECTRON MICROSCOPYf_plane_restr0.0082363

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