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Yorodumi- EMDB-5665: Cryo-EM structure of beta-hydroxyhexaketide-PikAIII conformation 1 -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5665 | |||||||||
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Title | Cryo-EM structure of beta-hydroxyhexaketide-PikAIII conformation 1 | |||||||||
Map data | Reconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide. | |||||||||
Sample |
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Keywords | Type I polyketide synthase module | |||||||||
Function / homology | Function and homology information 10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / acyltransferase activity, transferring groups other than amino-acyl groups / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / identical protein binding ...10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / acyltransferase activity, transferring groups other than amino-acyl groups / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / identical protein binding / plasma membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | Streptomyces venezuelae (bacteria) / unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 10.7 Å | |||||||||
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 / Abstract: The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical ...The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5665.map.gz | 14.7 MB | EMDB map data format | |
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Header (meta data) | emd-5665-v30.xml emd-5665.xml | 13.5 KB 13.5 KB | Display Display | EMDB header |
Images | 400_5665.gif 80_5665.gif | 34.4 KB 2.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5665 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5665 | HTTPS FTP |
-Validation report
Summary document | emd_5665_validation.pdf.gz | 78.5 KB | Display | EMDB validaton report |
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Full document | emd_5665_full_validation.pdf.gz | 77.7 KB | Display | |
Data in XML | emd_5665_validation.xml.gz | 495 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5665 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5665 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5665.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.24 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-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 |
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Components |
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-Supramolecule #1000: The 5th module from the pikromycin biosynthetic pathway (PikAIII)...
Supramolecule | Name: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide type: sample / ID: 1000 Details: Sample was not frozen prior to loading on the grid. The sample was monodisperse. Oligomeric state: Dimer / Number unique components: 4 |
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Molecular weight | Experimental: 328 KDa / Theoretical: 328 KDa / Method: Gel filtration chromatography |
-Macromolecule #1: PikAIII
Macromolecule | Name: PikAIII / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes |
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Source (natural) | Organism: Streptomyces venezuelae (bacteria) |
Molecular weight | Experimental: 328 KDa / Theoretical: 328 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET28b |
Sequence | UniProtKB: Narbonolide/10-deoxymethynolide synthase PikA3, module 5 |
-Macromolecule #2: NADPH
Macromolecule | Name: NADPH / type: ligand / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: unidentified (others) |
-Macromolecule #3: Methylmalonyl-CoA
Macromolecule | Name: Methylmalonyl-CoA / type: ligand / ID: 3 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: unidentified (others) |
-Macromolecule #4: Thiophenol-pentaketide
Macromolecule | Name: Thiophenol-pentaketide / type: ligand / ID: 4 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: unidentified (others) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7.4 / Details: 50 mM HEPES, 100mM NaCl |
Grid | Details: Glow-discharged Quantifoil R2/200 mesh grid |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 89 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1.5-2.0 seconds before plunging. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Min: 89 K / Max: 89 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification. |
Date | Mar 9, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 462 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 66964 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: OTHER |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | The particles were selected manually. |
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CTF correction | Details: Each micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1, EMAN2 / Number images used: 24773 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 3
Initial model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |