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- EMDB-8690: IMPDH Filament in the GTP-bound collapsed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-8690
TitleIMPDH Filament in the GTP-bound collapsed conformation
Map dataIMPDH filament collapsed conformation
Sample
  • Complex: IMPDH filament, collapsed conformation
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / negative staining / Resolution: 25.0 Å
AuthorsKollman JM / Burrell AL / Johnson MC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM118396 United States
CitationJournal: Mol Biol Cell / Year: 2017
Title: Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.
Authors: Sajitha A Anthony / Anika L Burrell / Matthew C Johnson / Krisna C Duong-Ly / Yin-Ming Kuo / Jacqueline C Simonet / Peter Michener / Andrew Andrews / Justin M Kollman / Jeffrey R Peterson /
Abstract: Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and ...Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.
History
DepositionApr 17, 2017-
Header (metadata) releaseMay 3, 2017-
Map releaseAug 23, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8690.png

Downloads & links

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Map

FileDownload / File: emd_8690.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIMPDH filament collapsed conformation
Voxel sizeX=Y=Z: 4.14 Å
Density
Contour LevelBy AUTHOR: 0.325 / Movie #1: 0.325
Minimum - Maximum-0.23251207 - 0.8471362
Average (Standard dev.)0.00075578416 (±0.10125507)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 529.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.144.144.14
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z529.920529.920529.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.2330.8470.001

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Supplemental data

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Sample components

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Entire : IMPDH filament, collapsed conformation

EntireName: IMPDH filament, collapsed conformation
Components
  • Complex: IMPDH filament, collapsed conformation

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Supramolecule #1: IMPDH filament, collapsed conformation

SupramoleculeName: IMPDH filament, collapsed conformation / type: complex / ID: 1 / Parent: 0 / Details: IMPDH assembled in the presence of ATP and GTP
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
Molecular weightTheoretical: 385 kDa/nm

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.6
StainingType: NEGATIVE / Material: uranyl formate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 0.5 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 2242 / Software - Name: EMAN Boxer
CTF correctionSoftware - Name: CTFFIND3
Startup modelType of model: OTHER / Details: featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER (ver. 9)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 93.9 Å
Applied symmetry - Helical parameters - Δ&Phi: 35.5 °
Applied symmetry - Helical parameters - Axial symmetry: D4 (2x4 fold dihedral)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2242

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