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- PDB-9ryk: Aquifex aeolicus lumazine synthase 11-pentamer cage in complex wi... -

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Basic information

Entry
Database: PDB / ID: 9ryk
TitleAquifex aeolicus lumazine synthase 11-pentamer cage in complex with riboflavin synthase trimer
Components
  • 6,7-dimethyl-8-ribityllumazine synthase
  • Riboflavin synthase
KeywordsBIOSYNTHETIC PROTEIN / protein cage / protein engineering / self-assembly / geometry / pentamer / encapsulation
Function / homology
Function and homology information


riboflavin synthase / riboflavin synthase activity / 6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / cytoplasm / cytosol
Similarity search - Function
Lumazine-binding protein / Lumazine-binding domain / Lumazine binding domain / Riboflavin synthase alpha chain lumazine-binding repeat profile. / Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase / ATP synthase subunit alpha, N-terminal domain-like superfamily / Riboflavin synthase-like beta-barrel
Similarity search - Domain/homology
6,7-dimethyl-8-ribityllumazine synthase / Riboflavin synthase
Similarity search - Component
Biological speciesAquifex aeolicus VF5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKoziej, L. / Azuma, Y.
Funding support Poland, European Union, 4items
OrganizationGrant numberCountry
Polish National Science Centre2020/37/B/NZ1/01187 Poland
Polish National Science Centre2018/31/D/NZ1/01102 Poland
Polish National Science Centre2019/35/B/NZ1/02044 Poland
European Molecular Biology Organization (EMBO)EMBO Installation Grant (YA)European Union
CitationJournal: Nat Commun / Year: 2026
Title: A molecular basis for stoichiometric enzyme encapsulation in the vitamin B2 biosynthesis compartment.
Authors: Lukasz Koziej / Jedrzej Pankowski / Monika Stefanska / Daniel Jankowski / Agnieszka Gawin / V Vishal Malolan / Juha T Huiskonen / Takahiro Kosugi / Yusuke Azuma /
Abstract: Encapsulating metabolic enzymes within protein cages enhances catalytic efficiency through substrate channeling. The vitamin B2 biosynthesis system, in which a dodecahedral lumazine synthase (LS) ...Encapsulating metabolic enzymes within protein cages enhances catalytic efficiency through substrate channeling. The vitamin B2 biosynthesis system, in which a dodecahedral lumazine synthase (LS) cage encapsulates a homotrimeric riboflavin synthase (RS), exemplifies this strategy, yet the molecular basis for this stoichiometric enzyme encapsulation has remained elusive. Here, cryogenic electron microscopy structures reveal a hierarchical assembly mechanism that ensures the defined host-guest ratio. RS C-terminal cage-localization signal peptides anchor at LS pentamer-pentamer interfaces early during assembly, stabilizing open intermediates that, together with delayed later-stage cage closure, extend the loading window until guest incorporation is complete. RS spatial occupancy avoids overloading, while a molecular lock upon final closure prevents disassembly. The elucidated anchoring mechanism enabled structure-based phylogenetic analysis across diverse organisms, suggesting multiple independent evolutionary origins of this modular encapsulation strategy. This naturally occurring architecture provides design principles for engineering synthetic catalytic compartments with programmable stoichiometric control.
History
DepositionJul 15, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Riboflavin synthase
2: Riboflavin synthase
3: Riboflavin synthase
A1: 6,7-dimethyl-8-ribityllumazine synthase
A2: 6,7-dimethyl-8-ribityllumazine synthase
A3: 6,7-dimethyl-8-ribityllumazine synthase
A4: 6,7-dimethyl-8-ribityllumazine synthase
A5: 6,7-dimethyl-8-ribityllumazine synthase
B1: 6,7-dimethyl-8-ribityllumazine synthase
B2: 6,7-dimethyl-8-ribityllumazine synthase
B3: 6,7-dimethyl-8-ribityllumazine synthase
B4: 6,7-dimethyl-8-ribityllumazine synthase
B5: 6,7-dimethyl-8-ribityllumazine synthase
C1: 6,7-dimethyl-8-ribityllumazine synthase
C2: 6,7-dimethyl-8-ribityllumazine synthase
C3: 6,7-dimethyl-8-ribityllumazine synthase
C4: 6,7-dimethyl-8-ribityllumazine synthase
C5: 6,7-dimethyl-8-ribityllumazine synthase
D1: 6,7-dimethyl-8-ribityllumazine synthase
D2: 6,7-dimethyl-8-ribityllumazine synthase
D3: 6,7-dimethyl-8-ribityllumazine synthase
D4: 6,7-dimethyl-8-ribityllumazine synthase
D5: 6,7-dimethyl-8-ribityllumazine synthase
E1: 6,7-dimethyl-8-ribityllumazine synthase
E2: 6,7-dimethyl-8-ribityllumazine synthase
E3: 6,7-dimethyl-8-ribityllumazine synthase
E4: 6,7-dimethyl-8-ribityllumazine synthase
E5: 6,7-dimethyl-8-ribityllumazine synthase
F1: 6,7-dimethyl-8-ribityllumazine synthase
F2: 6,7-dimethyl-8-ribityllumazine synthase
F3: 6,7-dimethyl-8-ribityllumazine synthase
F4: 6,7-dimethyl-8-ribityllumazine synthase
F5: 6,7-dimethyl-8-ribityllumazine synthase
G1: 6,7-dimethyl-8-ribityllumazine synthase
G2: 6,7-dimethyl-8-ribityllumazine synthase
G3: 6,7-dimethyl-8-ribityllumazine synthase
G4: 6,7-dimethyl-8-ribityllumazine synthase
G5: 6,7-dimethyl-8-ribityllumazine synthase
H1: 6,7-dimethyl-8-ribityllumazine synthase
H2: 6,7-dimethyl-8-ribityllumazine synthase
H3: 6,7-dimethyl-8-ribityllumazine synthase
H4: 6,7-dimethyl-8-ribityllumazine synthase
H5: 6,7-dimethyl-8-ribityllumazine synthase
I1: 6,7-dimethyl-8-ribityllumazine synthase
I2: 6,7-dimethyl-8-ribityllumazine synthase
I3: 6,7-dimethyl-8-ribityllumazine synthase
I4: 6,7-dimethyl-8-ribityllumazine synthase
I5: 6,7-dimethyl-8-ribityllumazine synthase
J1: 6,7-dimethyl-8-ribityllumazine synthase
J2: 6,7-dimethyl-8-ribityllumazine synthase
J3: 6,7-dimethyl-8-ribityllumazine synthase
J4: 6,7-dimethyl-8-ribityllumazine synthase
J5: 6,7-dimethyl-8-ribityllumazine synthase
K1: 6,7-dimethyl-8-ribityllumazine synthase
K2: 6,7-dimethyl-8-ribityllumazine synthase
K3: 6,7-dimethyl-8-ribityllumazine synthase
K4: 6,7-dimethyl-8-ribityllumazine synthase
K5: 6,7-dimethyl-8-ribityllumazine synthase


Theoretical massNumber of molelcules
Total (without water)1,066,65358
Polymers1,066,65358
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Riboflavin synthase / RS


Mass: 23227.842 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Gene: ribE, ribC, aq_1707 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-Gold(DE3) / References: UniProt: O67604, riboflavin synthase
#2: Protein ...
6,7-dimethyl-8-ribityllumazine synthase / DMRL synthase / LS / Lumazine synthase


Mass: 18126.709 Da / Num. of mol.: 55
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Gene: ribH, aq_132 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-Gold(DE3)
References: UniProt: O66529, 6,7-dimethyl-8-ribityllumazine synthase
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Aquifex aeolicus lumazine synthase 11-pentamer cage in complex with riboflavin synthase trimer
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.07 MDa / Experimental value: YES
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-Gold(DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium phosphateNaPi1
2200 mMsodium chlorideNaCl1
35 mMethylenediaminetetraacetic acidEDTA1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8625
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2EPU2.10.0.1941RELimage acquisition
4cryoSPARC4.5.3CTF correction
7UCSF ChimeraX1.9model fitting
8ISOLDE1.9model fitting
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
14PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1749455 / Details: 3D template picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25459 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial fitting was done using ChimeraX. Flexible fitting was done using Isolde.
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetails (eV)Initial refinement model-IDSource nameType
11HQK

1hqk

1HQKlumazine synthase1PDBexperimental model
2riboflavin synthaseAlphaFoldin silico model

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