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- PDB-9f48: KS + AT di-domain of polyketide synthase 13 in Mycobacterium tube... -

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Basic information

Entry
Database: PDB / ID: 9f48
TitleKS + AT di-domain of polyketide synthase 13 in Mycobacterium tuberculosis
ComponentsPolyketide synthase Pks13
KeywordsBIOSYNTHETIC PROTEIN / Mycolic acid synthesis / Claisen condensation / cell wall synthesis
Function / homology
Function and homology information


DIM/DIP cell wall layer assembly / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / fatty acid biosynthetic process / cytoplasm
Similarity search - Function
: / Thioesterase / Thioesterase domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. ...: / Thioesterase / Thioesterase domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Beta-ketoacyl synthase / 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 / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Polyketide synthase Pks13
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsJohnston, H.E. / Futterer, K.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/S000542/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R001154/1 United Kingdom
CitationJournal: Microbiology (Reading) / Year: 2024
Title: Cryo-electron microscopy structure of the di-domain core of polyketide synthase 13, essential for mycobacterial mycolic acid synthesis.
Authors: Hannah E Johnston / Sarah M Batt / Alistair K Brown / Christos G Savva / Gurdyal S Besra / Klaus Fütterer /
Abstract: Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. ...Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of , the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.
History
DepositionApr 26, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polyketide synthase Pks13
B: Polyketide synthase Pks13


Theoretical massNumber of molelcules
Total (without water)373,2842
Polymers373,2842
Non-polymers00
Water00
1


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

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Components

#1: Protein Polyketide synthase Pks13


Mass: 186642.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: pks13, Rv3800c / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: I6X8D2, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Has protein modificationN

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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: Subunit of Pks13 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.187 MDa / Experimental value: YES
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: H37Rv
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41(DE3)
Buffer solutionpH: 7.9
Details: 20 mM Tris-HCl pH 7.9, 50 mM NaCl, 2.5 mM beta-mercaptoethanol
Buffer componentConc.: 20 mM / Name: Tris / Formula: C4H11NO3
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 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: FLOOD BEAM
Electron lensMode: OTHER / Nominal defocus max: 2700 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 16.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1412 / Details: movie mode

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 310055
3D reconstructionResolution: 3.4 Å / Resolution method: OTHER / Num. of particles: 168566 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 70.54 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003114099
ELECTRON MICROSCOPYf_angle_d0.577119190
ELECTRON MICROSCOPYf_chiral_restr0.04232172
ELECTRON MICROSCOPYf_plane_restr0.00512533
ELECTRON MICROSCOPYf_dihedral_angle_d5.87062023

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