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- EMDB-2070: Octameric structure of IMPDH from Pseudomonas aeruginosa in prese... -

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Basic information

Entry
Database: EMDB / ID: EMD-2070
TitleOctameric structure of IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP
Map dataCryo-EM reconstruction of IMPDH from Pseudomonas Aeruginosa in presence of Mg-ATP
Sample
  • Sample: IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP filtered at 16A resolution
  • Protein or peptide: Inosine-5'-monophosphate dehydrogenase
KeywordsIMPDH / nucleotide metabolism / CBS domains
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsLabesse G / Vaupre L / alard-Arnaud I / Alexandre T / Lai Kee Him J / Raynal B / Bron P / Munier-Lehmann H
CitationJournal: Structure / Year: 2013
Title: MgATP regulates allostery and fiber formation in IMPDHs.
Authors: Gilles Labesse / Thomas Alexandre / Laurène Vaupré / Isabelle Salard-Arnaud / Joséphine Lai Kee Him / Bertrand Raynal / Patrick Bron / Hélène Munier-Lehmann /
Abstract: Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in nucleotide biosynthesis studied as an important therapeutic target and its complex functioning in vivo is still puzzling ...Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in nucleotide biosynthesis studied as an important therapeutic target and its complex functioning in vivo is still puzzling and debated. Here, we highlight the structural basis for the regulation of IMPDHs by MgATP. Our results demonstrate the essential role of the CBS tandem, conserved among almost all IMPDHs. We found that Pseudomonas aeruginosa IMPDH is an octameric enzyme allosterically regulated by MgATP and showed that this octameric organization is widely conserved in the crystal structures of other IMPDHs. We also demonstrated that human IMPDH1 adopts two types of complementary octamers that can pile up into isolated fibers in the presence of MgATP. The aggregation of such fibers in the autosomal dominant mutant, D226N, could explain the onset of the retinopathy adRP10. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or macromolecular assembly.
History
DepositionApr 6, 2012-
Header (metadata) releaseOct 3, 2012-
Map releaseApr 17, 2013-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 52
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 52
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2070.png

Downloads & links

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Map

FileDownload / File: emd_2070.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of IMPDH from Pseudomonas Aeruginosa in presence of Mg-ATP
Voxel sizeX=Y=Z: 2.3 Å
Density
Contour LevelBy AUTHOR: 52.0 / Movie #1: 52
Minimum - Maximum0.0 - 100.0
Average (Standard dev.)41.43122864 (±4.15516424)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 345.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.32.32.3
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z345.000345.000345.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean0.000100.00041.431

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Supplemental data

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Sample components

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Entire : IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP filtered ...

EntireName: IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP filtered at 16A resolution
Components
  • Sample: IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP filtered at 16A resolution
  • Protein or peptide: Inosine-5'-monophosphate dehydrogenase

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Supramolecule #1000: IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP filtered ...

SupramoleculeName: IMPDH from Pseudomonas aeruginosa in presence of Mg-ATP filtered at 16A resolution
type: sample / ID: 1000 / Oligomeric state: octameric / Number unique components: 1
Molecular weightTheoretical: 430 KDa

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Macromolecule #1: Inosine-5'-monophosphate dehydrogenase

MacromoleculeName: Inosine-5'-monophosphate dehydrogenase / type: protein_or_peptide / ID: 1 / Name.synonym: IMPDH / Number of copies: 8 / Oligomeric state: octamer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 430 KDa
Recombinant expressionOrganism: Bacteria (eubacteria)

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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

Concentration1 mg/mL
BufferpH: 8 / Details: 20 mM potassium phosphate pH 8 and 0.1 M KCl
GridDetails: Quantifoil R2.2, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 102.15 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 1 second

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 130137 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 100000
Specialist opticsEnergy filter - Name: JEOL / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: 626 Gatan cryo-Holder / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93.15 K / Max: 96.15 K / Average: 95.15 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
DetailsImage acquisition is done directly on the CCD camera. It explains why the nominal magnification is 100000 and calibrated magnification 130137.
DateNov 1, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 80 / Average electron dose: 18 e/Å2 / Details: Images directly recorded with CCD camera

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, FREALIGN / Number images used: 22577
DetailsThe particles were selected using a semi-automatic selection program (Boxer).

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