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- PDB-9pc6: Antibody (1B2) Bound Crosslinked Rifamycin Synthetase Module 1 wi... -
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Basic information
Entry | Database: PDB / ID: 9pc6 | ||||||
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Title | Antibody (1B2) Bound Crosslinked Rifamycin Synthetase Module 1 with a C-terminal Type II Thioesterase | ||||||
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![]() | Transferase/Immune System / Polyketide Synthase Module / Antibody (Fab) / Transferase-Immune System complex | ||||||
Function / homology | ![]() 6-deoxyerythronolide-B synthase / fatty acid synthase activity / lipid biosynthetic process / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / antibiotic biosynthetic process / fatty acid biosynthetic process / hydrolase activity Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.96 Å | ||||||
![]() | Cogan, D.P. / Liu, C. / West, R.C. / Chen, M. | ||||||
Funding support | 1items
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![]() | ![]() 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. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 659.7 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.6 MB | Display | ![]() |
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Full document | ![]() | 1.7 MB | Display | |
Data in XML | ![]() | 121.3 KB | Display | |
Data in CIF | ![]() | 180.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 71497MC ![]() 9patC ![]() 9pavC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 196008.141 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: O54666, UniProt: Q7BUF9, 6-deoxyerythronolide-B synthase #2: Antibody | Mass: 26447.611 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: Antibody | Mass: 25715.832 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Has ligand of interest | Y | Has protein modification | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Two antibody fragments (Fabs) in complex with homodimeric, crosslinked polyketide synthase module 1 of the rifamycin synthetase fused with a C-terminal type II thioesterase Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.49504131 MDa / Experimental value: YES | |||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||
Buffer solution | pH: 7.2 / Details: 100 mM citric acid, 10 mM HEPES, pH 7.2 (NaOH) | |||||||||||||||
Buffer component |
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Specimen | Conc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
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Electron microscopy imaging
Microscopy | Model: TFS GLACIOS |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3677 nm / Nominal defocus min: 100 nm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7631 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1229709 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91575 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model | ||||||||||||||||||||||||||||||||||||
Refinement | Highest resolution: 3.96 Å Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS) | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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