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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/homologyCTP synthase / CTP synthase / CTP synthase GATase domain / CTP synthase activity / CTP synthase, N-terminal / CTP synthase N-terminus / 'de novo' CTP biosynthetic process / Glutamine amidotransferase / Glutamine amidotransferase type 1 domain profile. / CTP biosynthetic process ...CTP synthase / CTP synthase / CTP synthase GATase domain / CTP synthase activity / CTP synthase, N-terminal / CTP synthase N-terminus / 'de novo' CTP biosynthetic process / Glutamine amidotransferase / Glutamine amidotransferase type 1 domain profile. / CTP biosynthetic process / Glutamine amidotransferase class-I / glutamine metabolic process / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase / ATP binding / identical protein binding / metal ion binding / cytosol / CTP synthase / CTP synthase
Function and homology information
Specimen sourceEscherichia coli / / bacteria /
MethodElectron microscopy (7.9 Å resolution / Particle / Single particle) / Transmission electron microscopy
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 22, 2016 / Release: Apr 26, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 26, 2017Structure modelrepositoryInitial release
1.1May 17, 2017Structure modelDatabase references
1.2Jun 21, 2017Structure modelDatabase referencescitation_citation.country / _citation.journal_volume / _citation.page_first / _citation.page_last

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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
Polyers241,7884
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)6260
ΔGint (kcal/M)-39
Surface area (Å2)85480

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Components

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


Mass: 60446.980 Da / Num. of mol.: 4 / Source: (gene. exp.) Escherichia coli / / bacteria / / Gene: pyrG, ECS88_3048 / Production host: Escherichia coli
References: UniProt:B7MLA1, UniProt:P0A7E5*PLUS, EC:6.3.4.2 (CTP synthase)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: E. coli CTP synthase tetramer / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli
Source (recombinant)Organism: Escherichia coli / Plasmid: pET22
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml
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
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD
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
4cctfindCTF CORRECTION
5RELIONCTF CORRECTION
11RELIONINITIAL EULER ASSIGNMENT
12RELIONFINAL EULER ASSIGNMENT
14RELIONRECONSTRUCTION
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: D2
3D reconstructionResolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 6407 / Symmetry type: POINT

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