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Yorodumi- PDB-7uk4: KS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound -
+Open data
-Basic information
Entry | Database: PDB / ID: 7uk4 | ||||||||||||
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Title | KS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound | ||||||||||||
Components | Polyketide synthase PKS13 | ||||||||||||
Keywords | BIOSYNTHETIC 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 | ||||||||||||
Biological species | Mycolicibacterium smegmatis MC2 155 (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.94 Å | ||||||||||||
Authors | Kim, S.K. / Dickinson, M.S. / Finer-Moore, J.S. / Rosenberg, O.S. / Stroud, R.M. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7uk4.cif.gz | 395.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7uk4.ent.gz | 297.3 KB | Display | PDB format |
PDBx/mmJSON format | 7uk4.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7uk4_validation.pdf.gz | 896.7 KB | Display | wwPDB validaton report |
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Full document | 7uk4_full_validation.pdf.gz | 902.6 KB | Display | |
Data in XML | 7uk4_validation.xml.gz | 62.9 KB | Display | |
Data in CIF | 7uk4_validation.cif.gz | 96.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/uk/7uk4 ftp://data.pdbj.org/pub/pdb/validation_reports/uk/7uk4 | HTTPS FTP |
-Related structure data
Related structure data | 26574MC 8cuyC 8cuzC 8cv0C 8cv1C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 | 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 / | Has ligand of interest | Y | Sequence details | The 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Molecular weight |
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Source (natural) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||||||
Vitrification | Instrument: 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN |
Image recording | Average 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 |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4400000 | |||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | |||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 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 building | Protocol: AB INITIO MODEL / Space: REAL | |||||||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 45.01 Å2 | |||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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