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- PDB-8uc3: Cryo-EM structure of the AlbAB cyclodipeptide oxidase enzyme filament -

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Basic information

Entry
Database: PDB / ID: 8uc3
TitleCryo-EM structure of the AlbAB cyclodipeptide oxidase enzyme filament
Components
  • Albonoursin synthase
  • Protein AlbB
KeywordsOXIDOREDUCTASE / cyclodipeptide oxidase / cyclic dipeptide oxidase / nitroreductase-like / enzyme filament / flavoenzyme
Function / homology
Function and homology information


albonoursin synthase / oxidoreductase activity, acting on the CH-CH group of donors, oxygen as acceptor / cytoplasm
Similarity search - Function
Protein of unknown function DUF6092 / Family of unknown function (DUF6092) / Nitroreductase / Nitroreductase family / Nitroreductase-like
Similarity search - Domain/homology
FLAVIN MONONUCLEOTIDE / Protein AlbB / Albonoursin synthase
Similarity search - Component
Biological speciesStreptomyces noursei ATCC 11455 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.78 Å
AuthorsAndreas, M.P. / Giessen, T.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133325 United States
CitationJournal: Nat Commun / Year: 2024
Title: Cyclodipeptide oxidase is an enzyme filament.
Authors: Michael P Andreas / Tobias W Giessen /
Abstract: Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the ...Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,β-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.
History
DepositionSep 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 15, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Albonoursin synthase
D: Protein AlbB
C: Protein AlbB
B: Albonoursin synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,2226
Polymers65,3094
Non-polymers9132
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Albonoursin synthase


Mass: 21071.307 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces noursei ATCC 11455 (bacteria)
Gene: albA / Production host: Streptomyces coelicolor A3(2) (bacteria) / Strain (production host): John Innes Centre M145 / References: UniProt: Q8GED9
#2: Protein Protein AlbB


Mass: 11583.143 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces noursei ATCC 11455 (bacteria)
Gene: albB / Production host: Streptomyces coelicolor A3(2) (bacteria) / Strain (production host): John Innes Centre M145 / References: UniProt: Q8GED8
#3: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: AlbAB cyclodipeptide oxidase enzyme filament / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Streptomyces noursei ATCC 11455 (bacteria)
Source (recombinant)Organism: Streptomyces coelicolor A3(2) (bacteria) / Strain: John Innes Centre M145
Buffer solutionpH: 7.5 / Details: 20 mM NaCl, 150 mM Tris pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTrisC4H11NO31
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid was glow discharged for 60 seconds at 5 mA under vacuum
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: Grid was plunge frozen into liquid ethane using the following parameters: blot force- 5, blot time 2 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.03063 sec. / Electron dose: 50.12 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3015
Image scansWidth: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2SerialEMimage acquisition
4cryoSPARC4.2.1CTF correction
7UCSF ChimeraX1.5model fitting
9cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.13D reconstruction
13PHENIX1.20.1-4487model refinement
14Coot0.9.8.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 119.969 ° / Axial rise/subunit: 46.063 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1202923
3D reconstructionResolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 795765
Details: Final reconstruction was generated by a masked local refinement of the helically refined map. The mask encompassed one dimer of AlbA and two surrounding dimers of AlbB.
Symmetry type: HELICAL
Atomic model buildingB value: 107.4 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: A model of AlbAB was created using AlphaFold2 using MMseqs2 via ColabFold. The model was then fit into the cryoEM density using ChimeraX. The model was then iteratively refined using Coot v ...Details: A model of AlbAB was created using AlphaFold2 using MMseqs2 via ColabFold. The model was then fit into the cryoEM density using ChimeraX. The model was then iteratively refined using Coot v 0.9.8.1 and real-space refinement in Phenix v 1.20.1-4487.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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