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- EMDB-61363: Structure of ATP-dependent diazotase, CmaA6 -

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Basic information

Entry
Database: EMDB / ID: EMD-61363
TitleStructure of ATP-dependent diazotase, CmaA6
Map data
Sample
  • Complex: Tetramer of CmaA6
    • Protein or peptide: ATP-dependent diazotase, CmaA6
Keywordsdiazotase / biosynthesis / LIGASE
Function / homologyCoA-ligase activity / ANL, N-terminal domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / AMP-dependent synthetase/ligase / AMP-binding enzyme / Putative AMP-binding enzyme
Function and homology information
Biological speciesKutzneria albida DSM 43870 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsKatsuyama Y / Kawai S / Ohnishi Y
Funding support Japan, 5 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19H04645 Japan
Japan Society for the Promotion of Science (JSPS)22H05130 Japan
Japan Society for the Promotion of Science (JSPS)19H05685 Japan
Japan Society for the Promotion of Science (JSPS)22KJ1046 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121001 Japan
CitationJournal: Angew Chem Int Ed Engl / Year: 2025
Title: Structural Basis for the Catalytic Mechanism of ATP-Dependent Diazotase CmaA6.
Authors: Seiji Kawai / Masayuki Karasawa / Yoshitaka Moriwaki / Tohru Terada / Yohei Katsuyama / Yasuo Ohnishi /
Abstract: Although several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP-dependent ...Although several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP-dependent diazotase, which catalyzes the diazotization of 3-aminocoumaric acid using nitrous acid. X-ray crystallography and cryogenic electron microscopy-single particle analysis revealed CmaA6 structures in the substrate-free and AMP-binding states. Kinetic analysis suggested that CmaA6 catalyzes diazotization via a sequential reaction mechanism in which three substrates (nitrous acid, ATP, and 3-aminocoumaric acid) are simultaneously bound in the reaction pocket. The nitrous acid and 3-aminocoumaric acid binding sites were predicted based on the AMP-binding state and confirmed by site-directed mutagenesis. In addition, computational analysis revealed a tunnel for 3-aminocoumaric acid to enter the reaction pocket, which was advantageous for the sequential reaction mechanism. This study provides important insights into the catalytic mechanism of diazotization in natural product biosynthesis.
History
DepositionAug 29, 2024-
Header (metadata) releaseMay 7, 2025-
Map releaseMay 7, 2025-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61363.png

Downloads & links

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Map

FileDownload / File: emd_61363.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 302.4 Å		0.84 Å/pix.
x 360 pix.
= 302.4 Å		0.84 Å/pix.
x 360 pix.
= 302.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.3336237 - 0.7300072
Average (Standard dev.)0.000269684 (±0.01924484)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61363_msk_1.map
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Half map: #1

Fileemd_61363_half_map_1.map
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Half map: #2

Fileemd_61363_half_map_2.map
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Sample components

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Entire : Tetramer of CmaA6

EntireName: Tetramer of CmaA6
Components
  • Complex: Tetramer of CmaA6
    • Protein or peptide: ATP-dependent diazotase, CmaA6

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Supramolecule #1: Tetramer of CmaA6

SupramoleculeName: Tetramer of CmaA6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Recombinant CmaA6 produced from Escherichia coli
Source (natural)Organism: Kutzneria albida DSM 43870 (bacteria)

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Macromolecule #1: ATP-dependent diazotase, CmaA6

MacromoleculeName: ATP-dependent diazotase, CmaA6 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Kutzneria albida DSM 43870 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: mnhkvhhhhh hiegrhmitk eeraqinadp elgagnvlhr lraygrptdr pvlwtdgtwr apdgshpev itlgelyeyv etyagfyhgk girprdvvgv ltasstefai nfmainslga i psfanakl rpeiareyir rqgasgavtd terhevlagg elgfvvtaed ...String:
mnhkvhhhhh hiegrhmitk eeraqinadp elgagnvlhr lraygrptdr pvlwtdgtwr apdgshpev itlgelyeyv etyagfyhgk girprdvvgv ltasstefai nfmainslga i psfanakl rpeiareyir rqgasgavtd terhevlagg elgfvvtaed irpehraqlp qg wpyrhdp tdpiiishss gttgmpkavp hthqtllyaq lhrlklsvgg smgrllvalp gnh naamsv mmfgllldsp vylqssqrgs dvldaiekfk pttvfgfsgt ygqiatsdls trdm ssiea yyntgdaahe ahirvlvaqg sheeigpdfk pvrvpgsvft dglgssetgy sifhn ghkp gsasfgrcig kpmsfaqaav lsedgrplpa gevgrlgvrs ptltpgywnd sltwhk lrl ggywltgdla mqdaegnfyh ldrapdairt eagivfstrt eelllaslpe ladctvt ai aeegvradwd gdgvaeayvl lqftdgarep gdltgwvnev lagqgfppvt ralrmdst d vstgvtgkvl krvmrerare lvaradqg

UniProtKB: Putative AMP-binding enzyme

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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

Concentration2.5 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
20.0 mMC4H12NO3CllTris-HCl
2.0 mMC9H9NO33-amino-4-coumaric acid
2.0 mMNaNO2sodium nitrite
2.0 mMMgCl2magnesium chloride
2.0 mMC10H16N5O13P3ATP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 5 sec. / Pretreatment - Pressure: 0.015 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2903985
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4) / Software - details: Patch CTF Estimation / Type: NONE
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Software - details: Non-uniform refinement / Number images used: 74530
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.4)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.4)
Final 3D classificationNumber classes: 1 / Avg.num./class: 74530 / Software - Name: cryoSPARC (ver. 4.4)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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