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- EMDB-39478: Structure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron ... -

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Basic information

Entry
Database: EMDB / ID: EMD-39478
TitleStructure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron Microscopy to 1.42 Angstrom Resolution
Map dataPost processed map
Sample
  • Complex: Aquifex aeolicus Lumazine Synthase
    • Protein or peptide: Lumazine synthase
KeywordsEnzyme involved in riboflavin biosynthesis / BIOSYNTHETIC PROTEIN
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 / cytoplasm
Similarity search - Function
Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Domain/homology
6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.42 Å
AuthorsSavva CG / Sobhy M / De Biasio A / Hamdan SM
Funding support1 items
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 16, 2024-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39478.png

Downloads & links

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Map

FileDownload / File: emd_39478.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost processed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.46 Å/pix.
x 700 pix.
= 318.71 Å		0.46 Å/pix.
x 700 pix.
= 318.71 Å		0.46 Å/pix.
x 700 pix.
= 318.71 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.4553 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.16591452 - 0.34502378
Average (Standard dev.)0.000018229632 (±0.0066114715)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions700700700
Spacing700700700
CellA=B=C: 318.71 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39478_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_39478_half_map_1.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_39478_half_map_2.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Aquifex aeolicus Lumazine Synthase

EntireName: Aquifex aeolicus Lumazine Synthase
Components
  • Complex: Aquifex aeolicus Lumazine Synthase
    • Protein or peptide: Lumazine synthase

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Supramolecule #1: Aquifex aeolicus Lumazine Synthase

SupramoleculeName: Aquifex aeolicus Lumazine Synthase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aquifex aeolicus (bacteria)
Molecular weightTheoretical: 1.065 MDa

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Macromolecule #1: Lumazine synthase

MacromoleculeName: Lumazine synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Aquifex aeolicus (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MQIYEGKLTA EGLRFGIVAS RFNHALVDRL VEGAIDCIVR HGGREEDITL VRVPGSWEIP VAAGELARK EDIDAVIAIG VLIRGATPHF DYIASEVSKG LANLSLELRK PITFGVITAD T LEQAIERA GTKHGNKGWE AALSAIEMAN LFKSLRWSHP QFEK

UniProtKB: 6,7-dimethyl-8-ribityllumazine synthase

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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

Concentration2.75 mg/mL
BufferpH: 7.5 / Details: 20mM Tris pH 7.5, 150mM NaCl and 1mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time: 3 sec Wait time: 0 sec.
DetailsIn buffer 20mM Tris pH 7.5, 150mM NaCl and 1mM DTT

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 12657 / Average exposure time: 3.0 sec. / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 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: 1.2 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 698248
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 1.42 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 470878
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-8yt4:
Structure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron Microscopy to 1.42 Angstrom Resolution

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