|Entry||Database: EMDB / ID: 5667|
|Title||Cryo-EM structure of beta-hydroxyhexaketide-PikAIII conformation 3|
|Map data||Reconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide.|
|Sample||The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide:|
PikAIII / (ligand) x 3
|Keywords||Type I polyketide synthase module|
|Function / homology||Polyketide synthase, acyl transferase domain / Malonyl-CoA ACP transacylase, ACP-binding / Acyl transferase domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase, N-terminal domain / ACP-like superfamily / Polyketide synthase, docking domain superfmaily / NAD(P)-binding domain superfamily / Ketoacyl-synthetase, C-terminal extension / Polyketide synthase, beta-ketoacyl synthase domain ...Polyketide synthase, acyl transferase domain / Malonyl-CoA ACP transacylase, ACP-binding / Acyl transferase domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase, N-terminal domain / ACP-like superfamily / Polyketide synthase, docking domain superfmaily / NAD(P)-binding domain superfamily / Ketoacyl-synthetase, C-terminal extension / Polyketide synthase, beta-ketoacyl synthase domain / Polyketide synthase, phosphopantetheine-binding domain / Erythronolide synthase docking / Beta-ketoacyl synthase, active site / Thiolase-like / Acyl transferase/acyl hydrolase/lysophospholipase / KR domain / Polyketide synthase, docking domain / Acyl transferase / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal / Polyketide synthase, ketoreductase domain / Phosphopantetheine binding ACP domain / Phosphopantetheine attachment site / Acyl transferase domain superfamily / Ketoacyl-synthetase C-terminal extension / Phosphopantetheine attachment site. / Beta-ketoacyl synthases active site. / Carrier protein (CP) domain profile. / Beta-ketoacyl synthase, C-terminal domain / 10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / transferase activity, transferring acyl groups other than amino-acyl groups / phosphopantetheine binding / identical protein binding / Narbonolide/10-deoxymethynolide synthase PikA3, module 5|
Function and homology information
|Source||Streptomyces venezuelae (bacteria) / unidentified (others)|
|Method||single particle reconstruction / cryo EM / 11.6 Å resolution|
|Authors||Whicher JR / Dutta S / Hansen DA / Hale WA / Chemler JA / Narayan AR / Hakansson K / Sherman DH / Smith JL / Skiniotis G|
|Citation||Journal: Nature / Year: 2014|
Title: Structural rearrangements of a polyketide synthase module during its catalytic cycle.
Authors: Jonathan R Whicher / Somnath Dutta / Douglas A Hansen / Wendi A Hale / Joseph A Chemler / Annie M Dosey / Alison R H Narayan / Kristina Håkansson / David H Sherman / Janet L Smith / Georgios Skiniotis
|Date||Deposition: May 2, 2013 / Header (metadata) release: Jul 3, 2013 / Map release: Jun 25, 2014 / Last update: Oct 22, 2014|
|Structure viewer||EM map: |
Downloads & links
|File||emd_5667.map.gz (map file in CCP4 format, 27649 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.24 Å|
CCP4 map header:
-Entire The 5th module from the pikromycin biosynthetic pathway (PikAIII)...
|Entire||Name: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide|
Details: Sample was not frozen prior to loading on the grid. The sample was monodisperse.
Number of components: 4 / Oligomeric State: Dimer
|Mass||Theoretical: 328 kDa / Experimental: 328 kDa / Measured by: Gel filtration chromatography|
-Component #1: protein, PikAIII
|Protein||Name: PikAIII / Oligomeric Details: Dimer / Number of Copies: 2 / Recombinant expression: Yes|
|Mass||Theoretical: 328 kDa / Experimental: 328 kDa|
|Source||Species: Streptomyces venezuelae (bacteria)|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Vector: pET28b|
|External references||UniProt: Narbonolide/10-deoxymethynolide synthase PikA3, module 5|
-Component #2: ligand, NADPH
|Ligand||Name: NADPHNicotinamide adenine dinucleotide phosphate / Number of Copies: 1 / Recombinant expression: No|
|Source||Species: unidentified (others)|
-Component #3: ligand, Methylmalonyl-CoA
|Ligand||Name: Methylmalonyl-CoA / Recombinant expression: No / Number of Copies: 1|
|Source||Species: unidentified (others)|
-Component #4: ligand, Thiophenol-pentaketide
|Ligand||Name: Thiophenol-pentaketide / Recombinant expression: No / Number of Copies: 1|
|Source||Species: unidentified (others)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.1 mg/ml / Buffer solution: 50 mM HEPES, 100mM NaCl / pH: 7.4|
|Support film||Glow-discharged Quantifoil R2/200 mesh grid|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 89 K / Humidity: 100 % / Method: Blot for 2 seconds before plunging.|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Oct 12, 2012|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 50000 X (nominal), 66964 X (calibrated)|
Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification.
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
|Specimen Holder||Model: OTHER / Temperature: K ( 89 - 89 K)|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 387|
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 18779 / Details: The particles were selected manually.|
|3D reconstruction||Algorithm: Cross-common lines / Software: EMAN1, EMAN2 / CTF correction: Each micrograph / Resolution: 11.6 Å / Resolution method: FSC 0.5|
-Atomic model buiding
|Modeling #1||Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL|
Input PDB model: 2HG4
Chain ID: A, B
|Modeling #2||Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL|
Input PDB model: 2FR0
|Modeling #3||Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL|
Input PDB model: 2JU1
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