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- PDB-8yt4: Structure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron ... -

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Basic information

Entry
Database: PDB / ID: 8yt4
TitleStructure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron Microscopy to 1.42 Angstrom Resolution
Components6,7-dimethyl-8-ribityllumazine synthase
KeywordsBIOSYNTHETIC PROTEIN / Enzyme involved in riboflavin biosynthesis
Function / homology
Function and homology information


6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / cytosol
Similarity search - Function
Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Domain/homology
PHOSPHATE ION / 6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.42 Å
AuthorsSavva, C.G. / Sobhy, M.A. / De Biasio, A. / Hamdan, S.M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: IUCrJ / Year: 2024
Title: Structure of Aquifex aeolicus lumazine synthase by cryo-electron microscopy to 1.42 Å resolution.
Authors: Christos G Savva / Mohamed A Sobhy / Alfredo De Biasio / Samir M Hamdan /
Abstract: Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which ...Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Å or better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42 Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48 h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for ∼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5 Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.
History
DepositionMar 24, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 11, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 6,7-dimethyl-8-ribityllumazine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,8642
Polymers17,7691
Non-polymers951
Water99155
1
A: 6,7-dimethyl-8-ribityllumazine synthase
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)1,071,859120
Polymers1,066,16160
Non-polymers5,69860
Water1,08160
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: 6,7-dimethyl-8-ribityllumazine synthase
hetero molecules
x 5


  • icosahedral pentamer
  • 89.3 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)89,32210
Polymers88,8475
Non-polymers4755
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: 6,7-dimethyl-8-ribityllumazine synthase
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 107 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)107,18612
Polymers106,6166
Non-polymers5706
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein 6,7-dimethyl-8-ribityllumazine synthase / DMRL synthase / LS / Lumazine synthase


Mass: 17769.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (bacteria) / Gene: ribH, aq_132 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: O66529, 6,7-dimethyl-8-ribityllumazine synthase
#2: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 55 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Lumazine synthase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.065 MDa / Experimental value: NO
Source (natural)Organism: Aquifex aeolicus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 20mM Tris pH7.5, 150mM NaCl, 1 mM DTT
SpecimenConc.: 2.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 3O mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 270000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 400 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 / Temperature (min): 85 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 46 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12657
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU3.5.1image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION5initial Euler assignment
10RELION5final Euler assignment
11RELION5classification
12RELION53D reconstruction
13REFMAC5.8.0405model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 698240
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 1.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 470878 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 1HQK

1hqk


Pdb chain-ID: A / Accession code: 1HQK / Source name: PDB / Type: experimental model
RefinementResolution: 1.42→1.42 Å / Cor.coef. Fo:Fc: 0.669 / SU B: 0.611 / SU ML: 0.023 / ESU R: 0.006
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.26343 --
obs0.26343 4472966 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 20.034 Å2
Refinement stepCycle: 1 / Total: 1225
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.0121187
ELECTRON MICROSCOPYr_bond_other_d00.016549
ELECTRON MICROSCOPYr_angle_refined_deg1.391.6331605
ELECTRON MICROSCOPYr_angle_other_deg0.51.5461205
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.6135152
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.66510
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.54510206
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0820.2187
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.021175
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02117
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it1.2791.714611
ELECTRON MICROSCOPYr_mcbond_other1.2451.699606
ELECTRON MICROSCOPYr_mcangle_it1.9173.079762
ELECTRON MICROSCOPYr_mcangle_other1.8933.061758
ELECTRON MICROSCOPYr_scbond_it3.1442.32576
ELECTRON MICROSCOPYr_scbond_other3.1442.326573
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other5.0754.005837
ELECTRON MICROSCOPYr_long_range_B_refined6.81323.925433
ELECTRON MICROSCOPYr_long_range_B_other6.79223.775388
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 1.4→1.436 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.397 330167 -
obs--100 %

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