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- EMDB-30440: Cryo-EM analysis of the nonribosomal peptide synthetase, FmoA3 -

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Basic information

Entry
Database: EMDB / ID: EMD-30440
TitleCryo-EM analysis of the nonribosomal peptide synthetase, FmoA3
Map data
Sample
  • Organelle or cellular component: Structure of nonribosomal peptide synthetase, FmoA3
    • Protein or peptide: FmoA3
Function / homology
Function and homology information


organic cyclic compound biosynthetic process / lipid biosynthetic process / phosphopantetheine binding / antibiotic biosynthetic process / catalytic activity
Similarity search - Function
AMP-binding / Condensation domain / Condensation domain / Amino acid adenylation domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain ...AMP-binding / Condensation domain / Condensation domain / Amino acid adenylation domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / AMP-dependent synthetase/ligase / AMP-binding enzyme, C-terminal domain superfamily / AMP-binding enzyme / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Phenyloxazoline synthase MbtB
Similarity search - Component
Biological speciesStreptomyces sp. Sp080513GE-23 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsSone K / Harada A / Kawai S / Urano N / Adachi N / Moriya T / Kawasaki M / Katsuyama Y / Senda T / Ohnishi Y
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19H04645 Japan
Japan Agency for Medical Research and Development (AMED)JP20am0101071 Japan
CitationJournal: 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
DepositionAug 11, 2020-
Header (metadata) releaseApr 14, 2021-
Map releaseApr 14, 2021-
UpdateJun 30, 2021-
Current statusJun 30, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30440.png

Downloads & links

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Map

FileDownload / File: emd_30440.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.10546345 - 0.18579815
Average (Standard dev.)1.6414006e-05 (±0.0033828495)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 422.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.880.880.88
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ220220220
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.1050.1860.000

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Supplemental data

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Half map: #1

Fileemd_30440_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30440_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of nonribosomal peptide synthetase, FmoA3

EntireName: Structure of nonribosomal peptide synthetase, FmoA3
Components
  • Organelle or cellular component: Structure of nonribosomal peptide synthetase, FmoA3
    • Protein or peptide: FmoA3

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Supramolecule #1: Structure of nonribosomal peptide synthetase, FmoA3

SupramoleculeName: Structure of nonribosomal peptide synthetase, FmoA3 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptomyces sp. Sp080513GE-23 (bacteria)
Molecular weightTheoretical: 120 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21 / Recombinant plasmid: pColdI

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Macromolecule #1: FmoA3

MacromoleculeName: FmoA3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces sp. Sp080513GE-23 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
1.0 mMC4H10O2S2DTT
GridModel: 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 15 seconds (blot force 25).
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm 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
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 819697
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER
Details: An ab initio model was generated using RELION3's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 60 A for use as an initial model for 3D classification.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 76171
FSC plot (resolution estimation)

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