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- EMDB-33041: Lumazine Synthase from Aquifex aeolicus -

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Basic information

Entry
Database: EMDB / ID: EMD-33041
TitleLumazine Synthase from Aquifex aeolicus
Map dataCryoEM map of Lumazine synthase from Aquifex aeolicus at 2.3 Angstrom resolution
Sample
  • Complex: CryoEM structure of Lumazine synthase composed of 60 identical subunits
    • Protein or peptide: 6,7-dimethyl-8-ribityllumazine synthaseLumazine synthase
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
6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria) / Aquifex aeolicus VF5 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.33 Å
AuthorsSobhy MA / Hamdan SM
Funding support Saudi Arabia, 1 items
OrganizationGrant numberCountry
Other privateURF/1/3764-01-01 Saudi Arabia
CitationJournal: PLoS One / Year: 2022
Title: Cryo-electron structures of the extreme thermostable enzymes Sulfur Oxygenase Reductase and Lumazine Synthase.
Authors: Mohamed A Sobhy / Lingyun Zhao / Dalaver Anjum / Ali Behzad / Masateru Takahashi / Muhammad Tehseen / Alfredo De Biasio / Rachid Sougrat / Samir Hamdan /
Abstract: Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous ...Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous environment. Here, we present high resolution cryo-EM structures of two thermostable enzymes that exhibit multimeric cage-like structures arranged into two different point-group symmetries. First, we determined the structure of the Sulfur Oxygenase Reductase (SOR) enzyme that catalyzes both the oxygenation and disproportionation of elemental sulfur in Archea and is composed of 24 homomeric units each of MW ≃ 35 kDa arranged in octahedral symmetry. The structure of SOR from Acidianus ambivalens (7X9W) was determined at 2.78 Å resolution. The active site of each subunit inside the central nanocompartment is composed of Fe3+ coordinated to two water molecules and the three amino acids (H86, H90 and E114). Second, we determined the structure of Lumazine Synthase (LS) from Aquifex aeolicus (7X7M) at 2.33 Å resolution. LS forms a cage-like structure consisting of 60 identical subunits each of MW ≃ 15 kDa arranged in a strict icosahedral symmetry. The LS subunits are interconnected by ion-pair network. Due to their thermostability and relatively easy purification scheme, both SOR and LS can serve as a model for the catalytic and structural characterization of biocatalysts as well as a benchmark for cryo-EM sample preparation, optimization of the acquisition parameters and 3D reconstruction.
History
DepositionMar 9, 2022-
Header (metadata) releaseOct 12, 2022-
Map releaseOct 12, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33041.png

Downloads & links

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Map

FileDownload / File: emd_33041.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of Lumazine synthase from Aquifex aeolicus at 2.3 Angstrom resolution
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.019
Minimum - Maximum-0.072622135 - 0.13465837
Average (Standard dev.)-2.654171e-05 (±0.0063850316)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 349.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map 2

Fileemd_33041_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_33041_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 : CryoEM structure of Lumazine synthase composed of 60 identical su...

EntireName: CryoEM structure of Lumazine synthase composed of 60 identical subunits
Components
  • Complex: CryoEM structure of Lumazine synthase composed of 60 identical subunits
    • Protein or peptide: 6,7-dimethyl-8-ribityllumazine synthaseLumazine synthase

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Supramolecule #1: CryoEM structure of Lumazine synthase composed of 60 identical su...

SupramoleculeName: CryoEM structure of Lumazine synthase composed of 60 identical subunits
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aquifex aeolicus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: 6,7-dimethyl-8-ribityllumazine synthase

MacromoleculeName: 6,7-dimethyl-8-ribityllumazine synthase / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO / EC number: 6,7-dimethyl-8-ribityllumazine synthase
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria) / Strain: VF5
Molecular weightTheoretical: 16.727201 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIYEGKLTA EGLRFGIVAS RFNHALVDRL VEGAIDCIVR HGGREEDITL VRVPGSWEIP VAAGELARKE DIDAVIAIGV LIRGATPHF DYIASEVSKG LANLSLELRK PITFGVITAD TLEQAIERAG TKHGNKGWEA ALSAIEMANL FKSLR

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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

Concentration6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTris.HClTris hydrochloride
150.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK I
Details: The purified protein was applied to the glow discharged grids inside the chamber, blotted for a duration of 1.5 s, and then plunge-frozen in liquid ethane cooled by liquid nitrogen..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 344110
Details: particles were selected by LoG picking from 1516 micrographs
Startup modelType of model: INSILICO MODEL / In silico model: Initial model was generated in Relion
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 103328
FSC plot (resolution estimation)

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