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- EMDB-47526: Cryo-EM structure of Burkholderia cenocepacia orotate phosphoribo... -

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Basic information

Entry
Database: EMDB / ID: EMD-47526
TitleCryo-EM structure of Burkholderia cenocepacia orotate phosphoribosyltransferase
Map dataPrimary map
Sample
  • Organelle or cellular component: Hexamer
    • Protein or peptide: Orotate phosphoribosyltransferase
  • Ligand: ACETATE ION
  • Ligand: water
Keywordsorotic acid phosphoribosyltransferase / TRANSFERASE
Function / homology
Function and homology information


orotate phosphoribosyltransferase / orotate phosphoribosyltransferase activity / pyrimidine nucleobase biosynthetic process / 'de novo' UMP biosynthetic process / magnesium ion binding
Similarity search - Function
Orotate phosphoribosyl transferase domain / Orotate phosphoribosyltransferase / Phosphoribosyl transferase domain / Phosphoribosyltransferase-like / Phosphoribosyltransferase domain
Similarity search - Domain/homology
Orotate phosphoribosyltransferase
Similarity search - Component
Biological speciesBurkholderia cenocepacia J2315 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsSharma N / French JB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM124898 United States
CitationJournal: Arch Biochem Biophys / Year: 2025
Title: Structural and kinetic analysis of distinct active and inactive states of Burkholderia cenocepacia orotate phosphoribosyltransferase.
Authors: Nandini Sharma / Zachary R Turlington / Sean P Zupko / Michael N Catoggio / Christine M Lukacs / Dmitry Serbzhinskiy / Jan Abendroth / Thomas E Edwards / Donald D Lorimer / George Barrera / ...Authors: Nandini Sharma / Zachary R Turlington / Sean P Zupko / Michael N Catoggio / Christine M Lukacs / Dmitry Serbzhinskiy / Jan Abendroth / Thomas E Edwards / Donald D Lorimer / George Barrera / Sydney Willis / Olive Beyer / Sarah Toay / Teng Da Li / Andrew T Torelli / Katherine A Hicks / Jarrod B French /
Abstract: Orotate phosphoribosyltransferase (OPRT) catalyzes the reaction that adds the pyrimidine base to the ribose in the penultimate step of the de novo biosynthesis of pyrimidine nucleotides. The OPRT ...Orotate phosphoribosyltransferase (OPRT) catalyzes the reaction that adds the pyrimidine base to the ribose in the penultimate step of the de novo biosynthesis of pyrimidine nucleotides. The OPRT structure consists of an obligate dimer, conserved throughout the phosphoribosyltransferase family. Here, we describe the structural characterization of Burkholderia cenocepacia OPRT (BcOPRT), both by X-ray crystallography and Cryo electron microscopy (Cryo-EM). While the known dimer is present in the structure of BcOPRT, a putative hexameric form was also observed by multiple methods. Analyses by chromatography, Cryo-EM, and kinetics indicate that both dimeric and hexameric forms of this enzyme are present together in solution. Comparison of the kinetics of the native protein and two variants, which were specifically designed to prevent hexamerization, reveal that only the hexameric form is enzymatically active. Collectively, these data suggest that BcOPRT may use oligomerization to control overall enzymatic activity, thus contributing to the local regulation of pyrimidine biosynthesis in this organism.
History
DepositionOct 28, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47526.png

Downloads & links

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Map

FileDownload / File: emd_47526.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 384 pix.
= 250.368 Å		0.65 Å/pix.
x 384 pix.
= 250.368 Å		0.65 Å/pix.
x 384 pix.
= 250.368 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.652 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.34
Minimum - Maximum-1.9944228 - 2.7169666
Average (Standard dev.)-0.00017406954 (±0.059182003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 250.36801 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEMhancer map

Fileemd_47526_additional_1.map
AnnotationDeepEMhancer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_47526_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_47526_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexamer

EntireName: Hexamer
Components
  • Organelle or cellular component: Hexamer
    • Protein or peptide: Orotate phosphoribosyltransferase
  • Ligand: ACETATE ION
  • Ligand: water

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Supramolecule #1: Hexamer

SupramoleculeName: Hexamer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Burkholderia cenocepacia J2315 (bacteria)
Molecular weightTheoretical: 26.56 kDa/nm

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Macromolecule #1: Orotate phosphoribosyltransferase

MacromoleculeName: Orotate phosphoribosyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: orotate phosphoribosyltransferase
Source (natural)Organism: Burkholderia cenocepacia J2315 (bacteria)
Molecular weightTheoretical: 26.561299 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHHHHHHMT GYDRQSISDT TAKILLEVQA VHFNAEKPFI FTSGWASPVY IDCRKLISYP RVRRALMEMA ETTITRDIGF EQIDAVAGG ETAGIPFAAW IADRMMVPMQ YVRKKPKGFG RNAQIEGHLE EGSRVLLVED LTTDSRSKIN FVNALRTAGA T VNHCFVLF ...String:
MAHHHHHHMT GYDRQSISDT TAKILLEVQA VHFNAEKPFI FTSGWASPVY IDCRKLISYP RVRRALMEMA ETTITRDIGF EQIDAVAGG ETAGIPFAAW IADRMMVPMQ YVRKKPKGFG RNAQIEGHLE EGSRVLLVED LTTDSRSKIN FVNALRTAGA T VNHCFVLF HYNIFKESVS VLKDIDVDLH ALATWWDVLR VAKASGYFET KTLDEVEKFL HAPAEWSAAH GGATAPKE

UniProtKB: Orotate phosphoribosyltransferase

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Macromolecule #2: ACETATE ION

MacromoleculeName: ACETATE ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: ACT
Molecular weightTheoretical: 59.044 Da
Chemical component information

ChemComp-ACT:
ACETATE ION

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 69 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4ohc

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 739734
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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