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- PDB-9pat: Antibody (1B2) Bound Rifamycin Synthetase Module 1 in the Transac... -

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Basic information

Entry
Database: PDB / ID: 9pat
TitleAntibody (1B2) Bound Rifamycin Synthetase Module 1 in the Transacylation Mode
Components
  • (6-deoxyerythronolide-B synthase) x 2
  • Antibody Fragment 1B2 Heavy Chain
  • Antibody Fragment 1B2 Light Chain
KeywordsTransferase/Immune System / Polyketide Synthase Module / Antibody (Fab) / Transferase-Immune System complex
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / antibiotic biosynthetic process / fatty acid biosynthetic process
Similarity search - Function
Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / Polyketide synthase, dehydratase domain / PKS_DH / PKS_PP_betabranch / : / Polyketide synthase dehydratase domain / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension ...Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / Polyketide synthase, dehydratase domain / PKS_DH / PKS_PP_betabranch / : / Polyketide synthase dehydratase domain / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Polyketide synthase, ketoreductase domain / KR domain / ANL, N-terminal domain / Malonyl-CoA ACP transacylase, ACP-binding / : / AMP-binding enzyme C-terminal domain / AMP-binding enzyme, C-terminal domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / AMP-dependent synthetase/ligase / AMP-binding enzyme / 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. / AMP-binding enzyme, C-terminal domain superfamily / 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
6-deoxyerythronolide-B synthase
Similarity search - Component
Biological speciesAmycolatopsis mediterranei (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsCogan, D.P. / Liu, C. / West, R.C. / Chen, M.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: bioRxiv / Year: 2025
Title: Molecular Basis for Asynchronous Chain Elongation During Rifamycin Antibiotic Biosynthesis.
Authors: Chengli Liu / Ryan C West / Muyuan Chen / Whitaker Cohn / George Wang / Aryan M Mandot / Selena Kim / Dillon P Cogan /
Abstract: The rifamycin synthetase (RIFS) from the bacterium is a large (3.5 MDa) multienzyme system that catalyzes over 40 chemical reactions to generate a complex precursor to the antibiotic rifamycin B. It ...The rifamycin synthetase (RIFS) from the bacterium is a large (3.5 MDa) multienzyme system that catalyzes over 40 chemical reactions to generate a complex precursor to the antibiotic rifamycin B. It is considered a hybrid enzymatic assembly line and consists of an N-terminal nonribosomal peptide synthetase loading module followed by a decamodular polyketide synthase (PKS). While the biosynthetic functions are known for each enzymatic domain of RIFS, structural and biochemical analyses of this system from purified components are relatively scarce. Here, we examine the biosynthetic mechanism of RIFS through complementary crosslinking, kinetic, and structural analyses of its first PKS module (M1). Thiol-selective crosslinking of M1 provided a plausible molecular basis for previously observed conformational asymmetry with respect to ketosynthase (KS)-substrate carrier protein (CP) interactions during polyketide chain elongation. Our data suggest that C-terminal dimeric interfaces-which are ubiquitous in bacterial PKSs-force their adjacent CP domains to co-migrate between two equivalent KS active site chambers. Cryogenic electron microscopy analysis of M1 further supported this observation while uncovering its unique architecture. Single-turnover kinetic analysis of M1 indicated that although removal of C-terminal dimeric interfaces supported 2-fold greater KS-CP interactions, it did not increase the partial product occupancy of the homodimeric protein. Our findings cast light on molecular details of natural antibiotic biosynthesis that will aid in the design of artificial megasynth(et)ases with untold product structures and bioactivities.
History
DepositionJun 25, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 6-deoxyerythronolide-B synthase
B: 6-deoxyerythronolide-B synthase
C: 6-deoxyerythronolide-B synthase
G: Antibody Fragment 1B2 Heavy Chain
H: Antibody Fragment 1B2 Heavy Chain
I: Antibody Fragment 1B2 Light Chain
L: Antibody Fragment 1B2 Light Chain


Theoretical massNumber of molelcules
Total (without water)630,7477
Polymers630,7477
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein 6-deoxyerythronolide-B synthase


Mass: 175359.844 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Amycolatopsis mediterranei (bacteria) / Strain: NRRL B-3240 / Gene: rifA / Plasmid: pDC43 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BAP1
References: UniProt: O54666, 6-deoxyerythronolide-B synthase
#2: Protein 6-deoxyerythronolide-B synthase


Mass: 175700.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Amycolatopsis mediterranei (bacteria) / Strain: NRRL B-3240 / Gene: rifA / Plasmid: pDC43 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BAP1
References: UniProt: O54666, 6-deoxyerythronolide-B synthase
#3: Antibody Antibody Fragment 1B2 Heavy Chain


Mass: 26447.611 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Antibody Antibody Fragment 1B2 Light Chain


Mass: 25715.832 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria)
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: Two antibody fragments (Fabs) in complex with homodimeric polyketide synthase module 1 of the rifamycin synthetase
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.45449469 MDa / Experimental value: YES
Source (natural)Organism: Amycolatopsis mediterranei (bacteria) / Strain: NRRL B-3240
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BAP1
Buffer solutionpH: 7.2 / Details: 100 mM citric acid, 10 mM HEPES, pH 7.2 (NaOH)
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMcitric acidC6H8O71
210 mMhepesC8H18N2O4S1
SpecimenConc.: 10 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 R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3988 nm / Nominal defocus min: 1188 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.72 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10005

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.0particle selection
4cryoSPARC4.7.0CTF correction
7UCSF ChimeraXmodel fitting
9PHENIX1.20.1_4487model refinement
10cryoSPARC4.7.0initial Euler assignment
11cryoSPARC4.7.0final Euler assignment
12RELION5classification
13cryoSPARC4.7.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3057376
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37950 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.96 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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