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- PDB-8czc: AT from first module of the pikromycin synthase -

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Basic information

Entry
Database: PDB / ID: 8czc
TitleAT from first module of the pikromycin synthase
ComponentsNarbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
KeywordsBIOSYNTHETIC PROTEIN / acyltransferase / methylmalonyl-specific / polyketide synthase
Function / homology
Function and homology information


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
: / : / Polyketide synthase dehydratase domain / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / PKS_PP_betabranch / Polyketide synthase dehydratase N-terminal domain / PKS_DH / Polyketide synthase, dehydratase domain / Polyketide synthase, dehydratase domain superfamily ...: / : / Polyketide synthase dehydratase domain / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / PKS_PP_betabranch / Polyketide synthase dehydratase N-terminal domain / PKS_DH / Polyketide synthase, dehydratase domain / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, ketoreductase domain / KR domain / Malonyl-CoA ACP transacylase, ACP-binding / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / PKS_KR / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase/acyl hydrolase/lysophospholipase / Ketosynthase family 3 (KS3) domain profile. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / 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
Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
Similarity search - Component
Biological speciesStreptomyces venezuelae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsKeatinge-Clay, A.T. / Dickinson, M.S. / Miyazawa, T. / McCool, R.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM106112 United States
Welch FoundationF-1712 United States
CitationJournal: Structure / Year: 2022
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
DepositionMay 24, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Sep 14, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2


Theoretical massNumber of molelcules
Total (without water)33,0111
Polymers33,0111
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, This AT is functional since the engineered polyketide synthase, Pik127, that relies on it synthesizes a triketide lactone product.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2 / Narbonolide/10-deoxymethynolide synthase PikAI / Pikromycin polyketide synthase component PikAI / ...Narbonolide/10-deoxymethynolide synthase PikAI / Pikromycin polyketide synthase component PikAI / Pikromycin PKS component PikAI / Type I modular polyketide synthase PikAI / PKS


Mass: 33011.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: First AT domain of pikromycin synthase / Source: (gene. exp.) Streptomyces venezuelae (bacteria) / Gene: pikAI / Production host: Escherichia coli (E. coli) / Strain (production host): K207-3
References: UniProt: Q9ZGI5, 10-deoxymethynolide synthase, narbonolide synthase

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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: 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: all / Source: RECOMBINANT
Molecular weightValue: 0.41 MDa / Experimental value: NO
Source (natural)Organism: Streptomyces venezuelae (bacteria) / Strain: 15439
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: K207-3
Buffer solutionpH: 7.5
Details: Buffer was made fresh because TCEP rapidly degrades in phosphate
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMpotassium phosphateK3PO41
20.5 mMTCEPC9H15O6P1
33 mMmethylmalonyl-CoAC25H40N7O19P3S1
41 mMNADPHC21H30N7O17P31
SpecimenConc.: 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 supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Details: Collected 4284 movies at 0 degrees tilt and 2912 movies at -30 degrees tilt
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansMovie frames/image: 100

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3.2.0CTF correction
7UCSF ChimeraX1.1.1model fitting
8PHENIX1.19.2-4158model fitting
10Coot0.8.9.2model refinement
11PHENIX1.19.2-4158model refinement
12cryoSPARC3.2.0initial Euler assignment
13cryoSPARC3.2.0final Euler assignment
14cryoSPARC3.2.0classification
15cryoSPARC3.2.03D reconstruction
CTF correctionDetails: CTF correction performed by cryosparc during reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6761206
Details: From blob picker, 6,761,206 particles were selected. Then, from template picker, 2,350,020 particles were selected.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184737
Details: Maps were density modified using Phenix to a calculated resolution of 2.77 Angstroms.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Phenix
Atomic model buildingPDB-ID: 2QO3

2qo3


Accession code: 2QO3 / Source name: PDB / Type: experimental model

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