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- EMDB-2700: Cryo-EM structure of the CTP synthetase filament -

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Basic information

Entry
Database: EMDB / ID: EMD-2700
TitleCryo-EM structure of the CTP synthetase filament
Map datareconstruction of CTP synthetase filament
Sample
  • Sample: CTP synthetase filament
  • Protein or peptide: CTP synthetase
Keywordsnucleotide biosynthesis / enzyme polymerization / enzyme regulation / CTP / UTP / ATP
Biological speciesEscherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 8.4 Å
AuthorsKollman JM / Charles EJ / Hansen JM
CitationJournal: Elife / Year: 2014
Title: Large-scale filament formation inhibits the activity of CTP synthetase.
Authors: Rachael M Barry / Anne-Florence Bitbol / Alexander Lorestani / Emeric J Charles / Chris H Habrian / Jesse M Hansen / Hsin-Jung Li / Enoch P Baldwin / Ned S Wingreen / Justin M Kollman / Zemer Gitai /
Abstract: CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in ...CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.
History
DepositionJul 7, 2014-
Header (metadata) releaseJul 23, 2014-
Map releaseJul 23, 2014-
UpdateAug 27, 2014-
Current statusAug 27, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.53
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.53
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.53
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2700-ctp...

Downloads & links

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Map

FileDownload / File: emd_2700.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of CTP synthetase filament
Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy AUTHOR: 0.53 / Movie #1: 0.53
Minimum - Maximum-2.38333011 - 3.16936803
Average (Standard dev.)-0.0128391 (±0.25320092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 393.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-2.3833.169-0.013

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Supplemental data

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Sample components

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Entire : CTP synthetase filament

EntireName: CTP synthetase filament
Components
  • Sample: CTP synthetase filament
  • Protein or peptide: CTP synthetase

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Supramolecule #1000: CTP synthetase filament

SupramoleculeName: CTP synthetase filament / type: sample / ID: 1000
Details: Filament assembled from CTP synthetase incubated with substrates.
Oligomeric state: helical polymer of CTP synthetase homotetramers
Number unique components: 1

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Macromolecule #1: CTP synthetase

MacromoleculeName: CTP synthetase / type: protein_or_peptide / ID: 1 / Name.synonym: CTP synthase, CtpS
Details: CtpS was incubated in the presence of substrates, and filament form as the product CTP builds up in the reaction
Number of copies: 4 / Oligomeric state: 2-2-2 tetramer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: cytoplasm
Molecular weightExperimental: 56 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.85 mg/mL
BufferpH: 7.8
Details: 50mM Tris pH 7.8, 10 mM MgCl2, 1mM UTP, 1mM ATP, 0.2 mM GTP, 5 mM glutamine
GridDetails: 200 mesh carbon grid with Quantifoil 2/2 support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: 3-6s blot time before plubging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 94000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 94000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 150,000x mag
DateNov 1, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3000 / Average electron dose: 25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 79.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 48.3 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: OTHER / Software - Name: SPIDER, hsearch_lorentz, EMAN1, ctffind
DetailsParticle alignment, 3-D reconstruction, and helical and local point group symmetrization were carried out in SPIDER, and hsearch_lorentz was used for helical symmetry parameter searches.

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Atomic model buiding 1

Initial modelPDB ID:

2ad5


Chain - Chain ID: A
SoftwareName: Chimera
DetailsDomains were fit independently using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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