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- EMDB-38405: Cryo-EM structure of colibactin assembly line polyketide synthase... -

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Basic information

Entry
Database: EMDB / ID: EMD-38405
TitleCryo-EM structure of colibactin assembly line polyketide synthase ClbC (ACP-bound state)
Map data
Sample
  • Complex: ACP-bound ClbC
    • Protein or peptide: Colibactin polyketide synthase ClbC
Keywordscolibactin / microbiome / polyketide synthase / colorectal cancer / NRPS-PKS hybrid / biosynthetic protein / TRANSFERASE
Function / homology
Function and homology information


DIM/DIP cell wall layer assembly / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / plasma membrane / cytoplasm
Similarity search - Function
Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / : / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / 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 ...Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / : / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / 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 / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Colibactin polyketide synthase ClbC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKim J / Kim M / Kang JY
Funding support Korea, Republic Of, 2 items
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, 2023-
Header (metadata) releaseMay 21, 2025-
Map releaseMay 21, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38405.png

Downloads & links

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Map

FileDownload / File: emd_38405.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 256 pix.
= 279.091 Å		1.09 Å/pix.
x 256 pix.
= 279.091 Å		1.09 Å/pix.
x 256 pix.
= 279.091 Å

Surface

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0902 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-2.269382 - 3.4125137
Average (Standard dev.)0.0013806439 (±0.059481885)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 279.0912 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_38405_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_38405_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : ACP-bound ClbC

EntireName: ACP-bound ClbC
Components
  • Complex: ACP-bound ClbC
    • Protein or peptide: Colibactin polyketide synthase ClbC

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Supramolecule #1: ACP-bound ClbC

SupramoleculeName: ACP-bound ClbC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Colibactin polyketide synthase ClbC

MacromoleculeName: Colibactin polyketide synthase ClbC / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 99.302961 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFELRR QALEYASEMN GMEIAIIGMA VRFPQSRTLH EFWHNIVQGK ECVTFFSEE ELLAEGVEQS TLDNPAYVRA KPYIEGICDF DAAFFGYSHK EAQTLDPKSR VLHEVAYHAL EDAGYAQRTS D LITGVFVG ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFELRR QALEYASEMN GMEIAIIGMA VRFPQSRTLH EFWHNIVQGK ECVTFFSEE ELLAEGVEQS TLDNPAYVRA KPYIEGICDF DAAFFGYSHK EAQTLDPKSR VLHEVAYHAL EDAGYAQRTS D LITGVFVG ASEDVDWLRR SLSQIGGDAL NRFESGIYGH KDLLAHLIAY SLNLNGPVYS LYTSCSTSLS ATHIACRSLL FG ECDLALA GGITIDLPQK SGYFCQQGMI HSTDGHCRPF DSQASGTLFG DGAGVVVLRR LEDALAAGDR IYAVIRGSAV NND GKQKIG FVAPGHEGQK AVICAACHLA EVSPESIGYV ETHGTGTRIG DPIEFAALTE AFDTSHRQYC ALGAVKANIG HTHA AAGVA GLIKTALVLH HRTIPPLANY QMPNSKLDLA HSPFYIPIQP QEWPASRMPP RAGVSSFGIG GTNVHMILEG LNPAV RDDH DQVRAPVFIP LSAPSFEQLD ELTQQLTPLL ATLDASTLAY TQQVARPVFD CRRVIQVEND GTQAMLASLD NLMPDA PWG LHCPDLRTTN DCTYAQWLAH SAHYQREATA LTALLDGMNI PPAYCHAETW AAQANSSLLI RGCQTIAALK TWMNLLP TL TLLSGAGTGL LPAAAASGMI ATQDVLHLLW EMEQKALHLW LPERHEPIPG YVLAWQGNPI TDAQRNDRGF WSEALLAD T RELGEGVHSI NWVRLPPEIR EDVDVLRYVA QLWCAGINVD WAVWYGTPLP QRGSASAYPF AHNHYPLPGR VMGSVETQP EAGPETHHPY QARPVLSVPF VAAHSRGMQY ITGLMELLLE ISPVGVDDDF FELGGHSLLV TQLTSRLERD FNVHIDLLTL MENPNPRNI YAHIAAQLGG EDNLEIACQ

UniProtKB: Colibactin polyketide synthase ClbC

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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

BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTristris
200.0 mMNaClsodium chloride
10.0 mMMgCl2magnesium chloride
1.0 mMTCEPTris(2-carboxyethyl)phosphine hydrochloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 2 / Number real images: 13070 / Average electron dose: 58.85 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.73 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6827074
CTF correctionSoftware - Name: cryoSPARC (ver. 3.0.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.1) / Number images used: 301978
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 147367 / Software - Name: cryoSPARC (ver. 3.0.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model / Details: apo-ClbC
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8xjt:
Cryo-EM structure of colibactin assembly line polyketide synthase ClbC (ACP-bound state)

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