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- PDB-5u05: Cryo-EM structure of the E. coli CTP synthase tetramer -

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Basic information

Entry
Database: PDB / ID: 5u05
TitleCryo-EM structure of the E. coli CTP synthase tetramer
ComponentsCTP synthaseCTP synthetase
KeywordsLIGASE / nucleotide metabolism / enzyme / active
Function / homology
Function and homology information


cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / glutamine metabolic process / magnesium ion binding / protein-containing complex / ATP binding ...cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / glutamine metabolic process / magnesium ion binding / protein-containing complex / ATP binding / metal ion binding / identical protein binding / cytosol
Similarity search - Function
CTP synthase / CTP synthase N-terminus / CTP synthase GATase domain / CTP synthase, N-terminal / Glutamine amidotransferase type 1 domain profile. / Glutamine amidotransferase class-I / Glutamine amidotransferase / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CTP synthase / CTP synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.9 Å
AuthorsLynch, E.M. / Hicks, D.R. / Kollman, J.M.
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Human CTP synthase filament structure reveals the active enzyme conformation.
Authors: Eric M Lynch / Derrick R Hicks / Matthew Shepherd / James A Endrizzi / Allison Maker / Jesse M Hansen / Rachael M Barry / Zemer Gitai / Enoch P Baldwin / Justin M Kollman /
Abstract: The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we ...The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.
History
DepositionNov 22, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2017Provider: repository / Type: Initial release
Revision 1.1May 17, 2017Group: Database references
Revision 1.2Jun 21, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_volume ..._citation.country / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: CTP synthase
B: CTP synthase
C: CTP synthase
D: CTP synthase


Theoretical massNumber of molelcules
Total (without water)241,7884
Polymers241,7884
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6260 Å2
ΔGint-39 kcal/mol
Surface area85480 Å2

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Components

#1: Protein
CTP synthase / CTP synthetase / Cytidine 5'-triphosphate synthase / Cytidine triphosphate synthetase / CTPS / UTP--ammonia ligase


Mass: 60446.980 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: pyrG, ECS88_3048 / Production host: Escherichia coli (E. coli)
References: UniProt: B7MLA1, UniProt: P0A7E5*PLUS, CTP synthase (glutamine hydrolysing)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E. coli CTP synthase tetramer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET22
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 50 mM HEPES potassium, pH 8.0, 10 mM magnesium chloride, 0.6 mM UTP, 1.5 mM AMP-PNP, 0.2 mM GTP, 10 mM glutamine
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 68 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1DoG Pickerparticle selection
4CTFFINDCTF correction
5RELIONCTF correction
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6407 / Symmetry type: POINT

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