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- EMDB-23587: Cryo-EM structure of the elongation module of the bacillamide NRP... -

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Basic information

Entry
Database: EMDB / ID: EMD-23587
TitleCryo-EM structure of the elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
Map dataEM map of the locally refined bacillamide NRPS, BmdB, complexed with the oxidase, BmdC
Sample
  • Complex: Elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
    • Protein or peptide: BmdC, Oxidase
    • Protein or peptide: BmdB, bacillamide NRPS
  • Ligand: FLAVIN MONONUCLEOTIDE
KeywordsNonribosomal peptide synthetases Oxidase Bacillamide synthetases / BIOSYNTHETIC PROTEIN
Biological speciesThermoactinomyces vulgaris (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSharon I / Strauss M / Schmeing TM
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: 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
DepositionMar 5, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ly4
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ly4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23587.png

Downloads & links

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Map

FileDownload / File: emd_23587.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map of the locally refined bacillamide NRPS, BmdB, complexed with the oxidase, BmdC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 480 pix.
= 547.2 Å		1.14 Å/pix.
x 480 pix.
= 547.2 Å		1.14 Å/pix.
x 480 pix.
= 547.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.058 / Movie #1: 0.2
Minimum - Maximum-0.2732265 - 0.88426125
Average (Standard dev.)-0.000091860995 (±0.011290918)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 547.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z547.200547.200547.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.2730.884-0.000

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Supplemental data

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Mask #1

Fileemd_23587_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of the locally refined bacillamide...

Fileemd_23587_half_map_1.map
AnnotationHalf map A of the locally refined bacillamide NRPS, BmdB, complexed with the oxidase, BmdC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of the locally refined bacillamide...

Fileemd_23587_half_map_2.map
AnnotationHalf map B of the locally refined bacillamide NRPS, BmdB, complexed with the oxidase, BmdC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Elongation module of the bacillamide NRPS, BmdB, in complex with ...

EntireName: Elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
Components
  • Complex: Elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
    • Protein or peptide: BmdC, Oxidase
    • Protein or peptide: BmdB, bacillamide NRPS
  • Ligand: FLAVIN MONONUCLEOTIDE

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Supramolecule #1: Elongation module of the bacillamide NRPS, BmdB, in complex with ...

SupramoleculeName: Elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Thermoactinomyces vulgaris (bacteria)

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Macromolecule #1: BmdC, Oxidase

MacromoleculeName: BmdC, Oxidase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermoactinomyces vulgaris (bacteria)
Molecular weightTheoretical: 38.177254 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMGEQIFYW SPAKHWRMSD EGVVIGESTY TGMILEWFPE FYFFAQTGVT INRLLERFSS GSEKEANEIL ELLIQDRVLV EGILPPREV FSPQEGLFVN PYSEQIRYSK EALDYYVSEQ LNRTHAACRS TKIQLETSGA LPDIIQKRRS CRRFDMKTPV S FATFSNLL ...String:
GAMGEQIFYW SPAKHWRMSD EGVVIGESTY TGMILEWFPE FYFFAQTGVT INRLLERFSS GSEKEANEIL ELLIQDRVLV EGILPPREV FSPQEGLFVN PYSEQIRYSK EALDYYVSEQ LNRTHAACRS TKIQLETSGA LPDIIQKRRS CRRFDMKTPV S FATFSNLL SSLKQRKEDK ILYNYASAGG LYPIDVFVYV KPRRVEGVKA GFYYFNPADH SLVLVNNIDQ VIKDDHELIN QD IFAQSAF SVYLVYNARA SMPKYGAAGY FYACIEAGII TATLNMVAED LNVGLCSIGH MNFEEIQTFL KLEDHQVILH AIE GGLKID GAAAENLYFQ

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Macromolecule #2: BmdB, bacillamide NRPS

MacromoleculeName: BmdB, bacillamide NRPS / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoactinomyces vulgaris (bacteria)
Molecular weightTheoretical: 117.373578 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMEADLSEP FSLTEVQTAY MLGRNPQFEL SGISPQTYFE YETELDIARL SRSFQKVIQR HPMLRAVILP EGKQQILRDV PEYEIEVES LVSMPPEKQA ARLREERSRM IDHVFPLGQW PLFELKAFQL QEHTYLLCFR YDALLMDGAS MNLVGQDLMH Y YHQPDAQL ...String:
GAMEADLSEP FSLTEVQTAY MLGRNPQFEL SGISPQTYFE YETELDIARL SRSFQKVIQR HPMLRAVILP EGKQQILRDV PEYEIEVES LVSMPPEKQA ARLREERSRM IDHVFPLGQW PLFELKAFQL QEHTYLLCFR YDALLMDGAS MNLVGQDLMH Y YHQPDAQL PPLSFTFQDY MHIYDDMKRG TEYETAKAYW TNKLPDFPPA PSLLLAKDPA EIGTPNFQSL TTIITKDKWL KL RRLAQDK QVTPSALLCT VYGEVLAFWS NQRRLAINLT VFNRYPVHDE VEQIVGDFTS LILLDMDMDQ KQPFFTKVEQ TQS TLLDGL EHRHYDGVEF IRDYTRYHQM RPKAVMPIVF TSMLAGAGAF AWEEIGSLRH IHARTPQVYL DNVVIEKNGE LLVS WNYVE ELFDAEVMES MFTQFVELLD QLVEQGDINP LRISQKDYAL IDQYNATAEP IPAATLHQLF IDQAQRTPDQ VAVVF EQEW LTYSELDQRS NQVARFLQSR GIGRGDRVGV LAKRQVETII NLMAVLKAGA AYVPIDPDHP YERQTYILEN SSCKIL LDS DLYETMEISS YADGDLTPVA EPEDTAYVIY TSGSTGRPKG VIITHQAASN TIQDINRKFE VNEEDRIIGI SSMCFDL SV YDIFGTLSAG ATLVMIRDPR DMRELVRTVE RRGITIWNTV PAIMDLALDH VGSHFENISL RLVLLSGDWI PLPLPAKI N RHFPVADVIS LGGATEASIW SIYWPIEQVE ANWKSIPYGK PLANQTYYVL NYDQKMCPVG VIGDLYIGGA GLAQGYLND DQKTKDAFIM HPEFGPIYKT GDCGRMRPEG YIEFLGRQDY QVKIQGYRVE LEEISHCLLT YPDVDQAVVI DQTDERGMKF LVGYVVAQQ EIDEKALRKH LMEHLPEYMI PAHLVHLEQL PLTPNGKLDR KALPVPKKQR NAEKFVAPQA GLEKILASVW Q EVLNVEQI GANDHFFALG GDSIKAIQVS ARLFVQGYHL DTKSLFEFPV LRDVARTIKK LAAAENLYFQ

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Macromolecule #3: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 2 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 109.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 122038
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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