+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-30440 | |||||||||
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Title | Cryo-EM analysis of the nonribosomal peptide synthetase, FmoA3 | |||||||||
Map data | ||||||||||
Sample |
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Function / homology | Function and homology information 2,3-dihydroxybenzoate-serine ligase activity / enterobactin synthetase complex / enterobactin biosynthetic process / amino acid activation for nonribosomal peptide biosynthetic process / phosphopantetheine binding / acyl carrier activity / nucleotide binding / cytoplasm Similarity search - Function | |||||||||
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
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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. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and ...Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses 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 activity. The collective data support a proposed catalytic cycle of FmoA3. | |||||||||
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_30440.map.gz | 391.4 MB | EMDB map data format | |
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Header (meta data) | emd-30440-v30.xml emd-30440.xml | 18.7 KB 18.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_30440_fsc.xml | 17 KB | Display | FSC data file |
Images | emd_30440.png | 125.5 KB | ||
Others | emd_30440_half_map_1.map.gz emd_30440_half_map_2.map.gz | 337.2 MB 337.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-30440 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-30440 | HTTPS FTP |
-Validation report
Summary document | emd_30440_validation.pdf.gz | 490.6 KB | Display | EMDB validaton report |
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Full document | emd_30440_full_validation.pdf.gz | 490.2 KB | Display | |
Data in XML | emd_30440_validation.xml.gz | 24.9 KB | Display | |
Data in CIF | emd_30440_validation.cif.gz | 32.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30440 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30440 | HTTPS FTP |
-Related structure data
Related structure data | 6ltaC 6ltbC 6ltcC 6ltdC C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-11059 (Title: Structural and functional analyses of the tridomain-nonribosomal peptide synthetase FmoA3 for 4-methyloxazoline ring formation Data size: 1.4 TB Data #1: Structural and functional analyses of the tridomain-nonribosomal peptide synthetase FmoA3 for 4-methyloxazoline ring formation [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_30440.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 0.88 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: #1
File | emd_30440_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_30440_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Structure of nonribosomal peptide synthetase, FmoA3
Entire | Name: Structure of nonribosomal peptide synthetase, FmoA3 |
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Components |
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-Supramolecule #1: Structure of nonribosomal peptide synthetase, FmoA3
Supramolecule | Name: Structure of nonribosomal peptide synthetase, FmoA3 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Streptomyces sp. Sp080513GE-23 (bacteria) |
Molecular weight | Theoretical: 120 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21 / Recombinant plasmid: pColdI |
-Macromolecule #1: FmoA3
Macromolecule | Name: FmoA3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Streptomyces sp. Sp080513GE-23 (bacteria) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MTISVPGHGT LGAPDGAAAP GGEAAELPPL VPEPGDAGQP FPLTPTQQAL WVGRADAVDL GDIGCYGYFE WERPELDLA RYRRAWERLV AHHPGLRTVV RPDGTQHVLE RPGPVPITVE DLRQDPDAVR RLEESRAERG H RALDPGTW PMFDLRVVLL SGRVRVQLGI ...String: MTISVPGHGT LGAPDGAAAP GGEAAELPPL VPEPGDAGQP FPLTPTQQAL WVGRADAVDL GDIGCYGYFE WERPELDLA RYRRAWERLV AHHPGLRTVV RPDGTQHVLE RPGPVPITVE DLRQDPDAVR RLEESRAERG H RALDPGTW PMFDLRVVLL SGRVRVQLGI DLQLMDASSL FLNLFSDLVT LYDDPDAALA SQKLAFRDFA RW LEEDVRG GARWRADWAY WQERLDGLPP APDLPAARYG AQGPGKFERC MVRCPAEEFA LLRERALAHG LTE TELLVG AFAEVLRGWS SDPAFTLNVP VFQRFDVPGI EDVIGDYTNP ILLEARPEGR TVAERIVALA ARLR ADTRH ASVNGVEVLR ELARRRGLAA AAMPVVVTSL LGLPSAARSI TEFGTEVHSI TQTPQVSLDF QIRPE DGEL RLVWDHRSGA FAPGVVEGAF EAFLDLVGRM LADEPGHGVW EAPFADMRSR RDRAVWNETN DTAEPV PAV LLQERFFAQA RRTPDAEAVV ASGLRLTYDE LARHAYRIGN TLRERGVRPG DLVGVVMEKG WEQYAAV YG ILAAGGAYLP IDAASPRGRV ARLLESAGAG IVLTQSRLRD ELDLPAGTTV LRADTDFETA STAPLTPV Q GPDDPAYVIY TSGSTGEPKG VVVAHRGVAN LVRDVRRRFA VTPADRLLAL SGLHFDASVY DVFGPLACG ATVVVPPPFR RAEPDVWAEL VRDERVTFWN SVPVLLELLV GEAESRDDRP LATLRLAVVS GDWIPLDLPG RARAQAPGL RVVGSGGPTE TICWSLFHPI DAVDPQWTSI PYGKPIANQR YYIVDRDLRP RPTWARGEMA V ASPLGLAL GYLNDPERTA AKFVTLPGTG ERAYLTGDFG RLLPDGGIEI LGREDFQVKV AGQRIELGEI EA LLHRADG VRAAVVTAPR SSADVVRLQA FVVPETGARL SADALREHLS AELPAAMVPA AIRLLPELPL TAN GKVDRL ALARLAAAPE EAPEPEARTD YAPRTDVGLL AELVAACVAE LLGLDEVPTT GNFFRLGGDS LSGT RLASR LQDLLGAPVP IRTVFGNPVL GDLASAIAGD PAAGPQAIRV ARLLHTLEEP DEKPGEKPDA EPAGE PDAG SRT |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 8 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use. | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 15 seconds (blot force 25). | |||||||||
Details | This sample was mono-disperse. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1886 / Average exposure time: 50.07 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |