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- PDB-9jjb: Class 1 state of the GfsA KSQ-ancestralAT chimeric didomain in co... -

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Basic information

Entry
Database: PDB / ID: 9jjb
TitleClass 1 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Components
  • Polyketide synthase
  • Polyketide synthase GfsA
KeywordsLYASE / decarboxylase / polyketide synthase
Function / homology
Function and homology information


Lyases; Carbon-carbon lyases; Carboxy-lyases / secondary metabolite biosynthetic process / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / antibiotic biosynthetic process / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / fatty acid biosynthetic process / lyase activity
Similarity search - Function
Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / PKS_PP_betabranch / : / Polyketide synthase dehydratase domain / : ...Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / PKS_PP_betabranch / : / Polyketide synthase dehydratase domain / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Polyketide synthase, ketoreductase domain / KR domain / 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 / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / PKS_KR / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / 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 / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Chem-9EF / Polyketide synthase GfsA
Similarity search - Component
Biological speciesStreptomyces graminofaciens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsChisuga, T. / Liao, Z. / Adachi, N. / Kawasaki, M. / Moriya, T. / Senda, T. / Kudo, F. / Eguchi, T. / Miyanaga, A.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H02911 Japan
Japan Society for the Promotion of Science (JSPS)23K19283 Japan
CitationJournal: Nat Commun / Year: 2025
Title: Ancestral sequence reconstruction as a tool for structural analysis of modular polyketide synthases.
Authors: Taichi Chisuga / Shota Takinami / Zengwei Liao / Masayuki Karasawa / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Toshiya Senda / Tohru Terada / Fumitaka Kudo / Tadashi Eguchi / Shogo ...Authors: Taichi Chisuga / Shota Takinami / Zengwei Liao / Masayuki Karasawa / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Toshiya Senda / Tohru Terada / Fumitaka Kudo / Tadashi Eguchi / Shogo Nakano / Sohei Ito / Akimasa Miyanaga /
Abstract: Modular polyketide synthases (PKSs) are large multi-domain enzymes critical for the biosynthesis of polyketide antibiotics. However, challenges with structural analysis limits our mechanistic ...Modular polyketide synthases (PKSs) are large multi-domain enzymes critical for the biosynthesis of polyketide antibiotics. However, challenges with structural analysis limits our mechanistic understanding of modular PKSs. In this report, we explore the potential of ancestral sequence reconstruction (ASR) for structure analysis of target proteins. As a model, we focus on the FD-891 PKS loading module composed of ketosynthase-like decarboxylase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains. We construct a KSAncAT chimeric didomain by replacing the native AT with an ancestral AT (AncAT) using ASR. After confirming that KSAncAT chimeric didomain retains similar enzymatic function to the native KSAT didomain, we successfully determine a high-resolution crystal structure of the KSAncAT chimeric didomain and cryo-EM structures of the KS-ACP complex. These cryo-EM structures, which could not be determined for the native protein, exemplify the utility of ASR to enable cryo-EM single-particle analysis. Our findings demonstrate that integrating ASR with structural analysis provides deeper mechanistic insight into modular PKSs. Furthermore, applying ASR to a partial region of the targeted multi-domain proteins could expand the potential of ASR and may serve as a valuable framework for investigating the structure and function of various multi-domain proteins.
History
DepositionSep 13, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Aug 13, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._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 GfsA
C: Polyketide synthase
B: Polyketide synthase GfsA
D: Polyketide synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,6226
Polymers146,8564
Non-polymers7672
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 GfsA / FD-891 synthase GfsA / loading and modules 1 to 4


Mass: 63183.352 Da / Num. of mol.: 2 / Mutation: Q197C
Source method: isolated from a genetically manipulated source
Details: GfsA KSQ-ancestralAT / Source: (gene. exp.) Streptomyces graminofaciens (bacteria) / Gene: gfsA / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: E0D202, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups, Lyases; Carbon-carbon lyases; Carboxy-lyases
#2: Protein Polyketide synthase / acyl carrier protein


Mass: 10244.458 Da / Num. of mol.: 2 / Fragment: ACPL
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces graminofaciens (bacteria) / Gene: gfsA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E0D202
#3: Chemical ChemComp-9EF / N-[2-(acetylamino)ethyl]-N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alaninamide


Mass: 383.335 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C13H26N3O8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: Class 1 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 216 kDa/nm / Experimental value: NO
Source (natural)Organism: Streptomyces graminofaciens (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
15 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES-NaOH1
250 mMsodium chlorideNaCl1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / 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: 291 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
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30382 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028430
ELECTRON MICROSCOPYf_angle_d0.4711460
ELECTRON MICROSCOPYf_dihedral_angle_d15.5953000
ELECTRON MICROSCOPYf_chiral_restr0.0391322
ELECTRON MICROSCOPYf_plane_restr0.0041498

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