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- PDB-7uk4: KS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound -

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Basic information

Entry
Database: PDB / ID: 7uk4
TitleKS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound
ComponentsPolyketide synthase PKS13
KeywordsBIOSYNTHETIC PROTEIN / mycolic acid synthesis / ketosynthase / acyltransferase / multi-domain assembly
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / erythronolide synthase activity / DIM/DIP cell wall layer assembly / fatty acid synthase activity / secondary metabolite biosynthetic process / phosphopantetheine binding / fatty acid biosynthetic process / plasma membrane / cytoplasm
Similarity search - Function
Thioesterase / Thioesterase domain / Malonyl-CoA ACP transacylase, ACP-binding / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase/acyl hydrolase/lysophospholipase ...Thioesterase / Thioesterase domain / Malonyl-CoA ACP transacylase, ACP-binding / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / 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 / 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
Unknown ligand / Polyketide synthase PKS13
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.94 Å
AuthorsKim, S.K. / Dickinson, M.S. / Finer-Moore, J.S. / Rosenberg, O.S. / Stroud, R.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI095208 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI128214 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM24485 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.
Authors: Sun Kyung Kim / Miles Sasha Dickinson / Janet Finer-Moore / Ziqiang Guan / Robyn M Kaake / Ignacia Echeverria / Jen Chen / Ernst H Pulido / Andrej Sali / Nevan J Krogan / Oren S Rosenberg / Robert M Stroud /
Abstract: The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug ...The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.
History
DepositionMar 31, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 29, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyketide synthase PKS13
B: Polyketide synthase PKS13
hetero molecules


Theoretical massNumber of molelcules
Total (without water)397,8924
Polymers397,2672
Non-polymers6252
Water7,026390
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Polyketide synthase PKS13


Mass: 198633.359 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: The gene for M. smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was ...Details: The gene for M. smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was cleaved to yield the full-length Pks13.
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155
References: UniProt: I7FMV0, 6-deoxyerythronolide-B synthase
#2: Chemical ChemComp-UNL / UNKNOWN LIGAND


Mass: 312.530 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: The fatty acid ligand designated as UNL (unknown ligand) is a divided population of fatty acids of formula C55H106O2 and C40H78O2 as confirmed by mass spectrometry.
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 390 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Sequence detailsThe gene for M. smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C- ...The gene for M. smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was cleaved to yield the full-length Pks13.

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Mycobacterial polyketide synthase 13COMPLEXThe gene for Mycobacterium smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was cleaved to yield the full-length Pks13.#10NATURAL
2Ketosynthase domainCOMPLEXLinked to acyl transferase domain at the C-terminus#11NATURAL
3Acyl transferase domainCOMPLEXlinked to ketosynthase domain at the N-terminus#11NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Mycolicibacterium smegmatis MC2 155 (bacteria)246196
32Mycolicibacterium smegmatis MC2 155 (bacteria)246196
43Mycolicibacterium smegmatis MC2 155 (bacteria)246196
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMTris1
2150 mMNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K
Details: Full length pks13 was concentrated to 1.5 mg mL-1 for cryo-EM grid preparation. 4.5 uL of sample was applied to freshly glow discharged holey carbon on gold R1.2/1.3 300 mesh Quantifoil ...Details: Full length pks13 was concentrated to 1.5 mg mL-1 for cryo-EM grid preparation. 4.5 uL of sample was applied to freshly glow discharged holey carbon on gold R1.2/1.3 300 mesh Quantifoil grids and blotted for 9 s with Whatman 1 filter paper at max humidity and 10oC in a FEI Mark IV Vitrobot, before vitrification in liquid nitrogen-cooled liquid ethane.

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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: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5.9 sec. / Electron dose: 45.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 7567

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4cryoSPARC2.12CTF correction
7Coot0.8.9.2model fitting
9ISOLDEmodel refinementapplication in ChimeraX
10PHENIX1.19.2model refinement
11cryoSPARC2.12initial Euler assignment
12cryoSPARC2.12final Euler assignment
13cryoSPARC2.12classification
14cryoSPARC2.123D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4400000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1200000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 45.01 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414381
ELECTRON MICROSCOPYf_angle_d0.58319506
ELECTRON MICROSCOPYf_dihedral_angle_d14.6825248
ELECTRON MICROSCOPYf_chiral_restr0.0452175
ELECTRON MICROSCOPYf_plane_restr0.0042578

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