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Open data
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Basic information
Entry | Database: PDB / ID: 7uwr | |||||||||
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Title | KSQ+AT from first module of the pikromycin synthase | |||||||||
![]() | Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2 | |||||||||
![]() | BIOSYNTHETIC PROTEIN / ketosynthase-like / acyltransferase / decarboxylation | |||||||||
Function / homology | ![]() 10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / acyltransferase activity, transferring groups other than amino-acyl groups / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / plasma membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å | |||||||||
![]() | Keatinge-Clay, A.T. / Dickinson, M.S. / Miyazawa, T. / McCool, R.S. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Priming enzymes from the pikromycin synthase reveal how assembly-line ketosynthases catalyze carbon-carbon chemistry. Authors: Miles S Dickinson / Takeshi Miyazawa / Ryan S McCool / Adrian T Keatinge-Clay / ![]() Abstract: The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups ...The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KS and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KS and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KS active site, while an AT-swapped version of Pik127 demonstrated that KS can also decarboxylate malonyl groups. Mechanisms for how KS and KS domains catalyze carbon-carbon chemistry are proposed. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 284.8 KB | Display | ![]() |
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PDB format | ![]() | 224.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 26839MC ![]() 8czcC 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: 91797.898 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: Q9ZGI5, 10-deoxymethynolide synthase, narbonolide synthase #2: Water | ChemComp-HOH / | |
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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: Pik127 / Type: COMPLEX Details: Polyketide synthase engineered from the first, second, and seventh modules of the pikromycin synthase as described in Miyazawa et al., 2021, Chem. Commun. 57:8762-8765 Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Value: 0.41 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: Buffer was made fresh because TCEP rapidly degrades in phosphate | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: This sample was monodisperse. It was incubated with its substrates, methylmalonyl-CoA and NADPH, for 30 min. at 25 C before freezing. | |||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/2 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K Details: Blotted for 3 seconds with a force of "1" before plunging. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS Details: Collected 4284 movies at 0 degrees tilt and 2912 movies at -30 degrees tilt |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 61728 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 5 sec. / Electron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7196 / Details: 20 frames per second |
Image scans | Movie frames/image: 100 |
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Processing
EM software |
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CTF correction | Details: CTF correction performed by cryosparc during reconstruction Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 6761206 Details: From blob picker, 6,761,206 particles were selected. Then, from template picker, 2,350,020 particles were selected. | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184737 / Algorithm: FOURIER SPACE / Details: Density modification in Phenix / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Phenix | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 2QO3 Accession code: 2QO3 / Source name: PDB / Type: experimental model |