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- PDB-6fik: ACP2 crosslinked to the KS of the loading/condensing region of th... -

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Basic information

Entry
Database: PDB / ID: 6fik
TitleACP2 crosslinked to the KS of the loading/condensing region of the CTB1 PKS
Components(Polyketide synthase) x 2
KeywordsBIOSYNTHETIC PROTEIN / NR-PKS / PKS / iPKS / iterative PKS / non-reducing / SAT / starter acyl / condensing / loading / polyketide / fungal / crosslink / ACP / acyl carrier / transferase
Function / homology
Function and homology information


phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / fatty acid biosynthetic process
Similarity search - Function
Polyketide product template domain / Polyketide synthase, thioesterase domain / Thioesterase / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / Thioesterase / Thioesterase domain / Polyketide synthase, dehydratase domain superfamily / Malonyl-CoA ACP transacylase, ACP-binding ...Polyketide product template domain / Polyketide synthase, thioesterase domain / Thioesterase / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / Thioesterase / Thioesterase domain / Polyketide synthase, dehydratase domain superfamily / Malonyl-CoA ACP transacylase, ACP-binding / 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 / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Non-reducing polyketide synthase CTB1
Similarity search - Component
Biological speciesCercospora nicotianae (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsHerbst, D.A. / Huitt-Roehl, C.R. / Jakob, R.P. / Townsend, C.A. / Maier, T.
Funding support Switzerland, United States, 5items
OrganizationGrant numberCountry
Swiss National Science Foundation159696 Switzerland
Swiss National Science Foundation145023 Switzerland
Swiss National Science Foundation164074 Switzerland
Swiss National Science Foundation138262 Switzerland
National Institutes of HealthES001670 United States
CitationJournal: Nat Chem Biol / Year: 2018
Title: The structural organization of substrate loading in iterative polyketide synthases.
Authors: Dominik A Herbst / Callie R Huitt-Roehl / Roman P Jakob / Jacob M Kravetz / Philip A Storm / Jamie R Alley / Craig A Townsend / Timm Maier /
Abstract: Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an ...Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.
History
DepositionJan 18, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 21, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 4, 2018Group: Data collection / Database references / Category: citation / Item: _citation.year
Revision 1.2Apr 11, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Apr 25, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Assembly

Deposited unit
A: Polyketide synthase
B: Polyketide synthase
C: Polyketide synthase


Theoretical massNumber of molelcules
Total (without water)290,4033
Polymers290,4033
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, gel filtration, homology, negative stain EM and crystallography
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15190 Å2
ΔGint-65 kcal/mol
Surface area91400 Å2
MethodPISA

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Components

#1: Protein Polyketide synthase /


Mass: 140255.000 Da / Num. of mol.: 2 / Mutation: C119A, T321A, S1010A
Source method: isolated from a genetically manipulated source
Details: C553 in chain A is crosslinked to S1816 in chain C / Source: (gene. exp.) Cercospora nicotianae (fungus) / Production host: Escherichia coli (E. coli) / References: UniProt: Q6DQW3
#2: Protein Polyketide synthase /


Mass: 9892.837 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: S1816 is crosslinked to C553 in chain A / Source: (gene. exp.) Cercospora nicotianae (fungus) / Production host: Escherichia coli (E. coli) / References: UniProt: Q6DQW3

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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: CTB1-SAT0-KS-MAT0=ACP2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Cercospora nicotianae (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(hydroxymethyl)aminomethan (TRIS)C4H11NO31
250 mMsodium chlorideNaClSodium chloride1
32.5 mMtris(2-carboxyethyl)phosphine (TCEP)C9H15O6P1
SpecimenConc.: 0.27 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The proteins have been crosslinked via site specific crosslinking.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil Lacey carbon grid
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 27707 X / Nominal defocus max: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 4500 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 41 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter upper: 20 eV / Energyfilter lower: 0 eV

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Processing

SoftwareName: PHENIX / Version: 1.10_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN22.11particle selectione2boxer.py
2DigitalMicrograph2.32image acquisition
4CTFFIND4.1CTF correction
5Gctf1.06CTF correction
13RELION2classification
14RELION23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25107 / Num. of class averages: 2 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00839483
ELECTRON MICROSCOPYf_angle_d1.28971372
ELECTRON MICROSCOPYf_dihedral_angle_d14.60815825
ELECTRON MICROSCOPYf_chiral_restr0.0673122
ELECTRON MICROSCOPYf_plane_restr0.0076214

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