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Yorodumi- PDB-7ly4: Cryo-EM structure of the elongation module of the bacillamide NRP... -
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-Basic information
Entry | Database: PDB / ID: 7ly4 | ||||||
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Title | Cryo-EM structure of the elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC | ||||||
Components |
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Keywords | BIOSYNTHETIC PROTEIN / Nonribosomal peptide synthetases Oxidase Bacillamide synthetases | ||||||
Function / homology | FLAVIN MONONUCLEOTIDE Function and homology information | ||||||
Biological species | Thermoactinomyces vulgaris (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||
Authors | Sharon, I. / Fortinez, C.M. / Schmeing, T.M. | ||||||
Funding support | Canada, 1items
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Citation | Journal: Nat Commun / Year: 2022 Title: Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module. Authors: Camille Marie Fortinez / Kristjan Bloudoff / Connor Harrigan / Itai Sharon / Mike Strauss / T Martin Schmeing / Abstract: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and ...Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS. | ||||||
History |
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-Structure visualization
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 7ly4.cif.gz | 309.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ly4.ent.gz | 244.3 KB | Display | PDB format |
PDBx/mmJSON format | 7ly4.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ly4_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7ly4_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7ly4_validation.xml.gz | 60.9 KB | Display | |
Data in CIF | 7ly4_validation.cif.gz | 90.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ly/7ly4 ftp://data.pdbj.org/pub/pdb/validation_reports/ly/7ly4 | HTTPS FTP |
-Related structure data
Related structure data | 23587MC 7ly5C 7ly6C 7ly7C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 38177.254 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermoactinomyces vulgaris (bacteria) / Production host: Escherichia coli (E. coli) #2: Protein | | Mass: 117373.578 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermoactinomyces vulgaris (bacteria) / Production host: Escherichia coli (E. coli) #3: Chemical | Has ligand of interest | Y | |
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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 | Name: Elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Source (natural) | Organism: Thermoactinomyces vulgaris (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: 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: OTHER |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 109 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122038 / Symmetry type: POINT |