|Entry||Database: EMDB / ID: EMD-30440|
|Title||Cryo-EM analysis of the nonribosomal peptide synthetase, FmoA3|
|Sample||Structure of nonribosomal peptide synthetase, FmoA3:|
|Biological species||Streptomyces sp. Sp080513GE-23 (bacteria)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.55 Å|
|Authors||Sone K / Harada A / Kawai S / Urano N / Adachi N / Moriya T / Kawasaki M / Katsuyama Y / Senda T / Ohnishi Y|
|Funding support|| Japan, 2 items |
|Citation||Journal: Angew Chem Int Ed Engl / Year: 2021|
Title: Structural and functional analyses of the tridomain-nonribosomal peptide synthetase FmoA3 for 4-methyloxazoline ring formation.
Authors: Yohei Katsuyama / Kaoru Sone / Ayaka Harada / Seiji Kawai / Naoki Urano / Naruhiko Adachi / Toshio Moriya / Masato Kawasaki / Kazuo Shin-Ya / Toshiya Senda / Yasuo Ohnishi /
Abstract: Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides . F moA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), ...Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides . F moA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It use s α - methyl- l -serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography ( apo -form, with adenylyl-imidodiphosphate and α -methyl- l -seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α -methyl- l -serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activit y. The collective data support a proposed catalytic cycle of FmoA3.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_30440.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 0.88 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Half map: #1
|Projections & Slices|
-Half map: #2
-Entire Structure of nonribosomal peptide synthetase, FmoA3
|Entire||Name: Structure of nonribosomal peptide synthetase, FmoA3 / Number of components: 2|
-Component #1: cellular-component, Structure of nonribosomal peptide synthetase,...
|Cellular-component||Name: Structure of nonribosomal peptide synthetase, FmoA3 / Recombinant expression: No|
|Mass||Theoretical: 120 kDa|
|Source||Species: Streptomyces sp. Sp080513GE-23 (bacteria)|
|Source (engineered)||Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pColdI / Strain: BL21|
-Component #2: protein, FmoA3
|Protein||Name: FmoA3 / Recombinant expression: No|
|Source||Species: Streptomyces sp. Sp080513GE-23 (bacteria)|
|Source (engineered)||Expression System: Escherichia coli BL21(DE3) (bacteria)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 8 mg/mL / pH: 8|
|Support film||The grid was washed by acetone prior to use.|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 291 K / Humidity: 100 % / Details: Blotting time was 15 seconds (blot force 25).|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Imaging||Microscope: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 120000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2500.0 nm|
|Specimen Holder||Model: OTHER|
|Camera||Detector: FEI FALCON III (4k x 4k)|
|Image acquisition||Number of digital images: 1886|
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