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- PDB-5u6r: E. coli CTP synthase CC mutant filament (product-bound) -

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Basic information

Entry
Database: PDB / ID: 5u6r
TitleE. coli CTP synthase CC mutant filament (product-bound)
ComponentsCTP synthaseCTP synthetase
KeywordsPROTEIN FIBRIL / LIGASE / nucleotide metabolism / metabolic filament
Function / homology
Function and homology information


cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / glutaminase activity / CTP biosynthetic process / glutamine metabolic process / protein homotetramerization / magnesium ion binding ...cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / glutaminase activity / CTP biosynthetic process / glutamine metabolic process / protein homotetramerization / magnesium ion binding / protein-containing complex / ATP binding / identical protein binding / metal ion binding / cytosol
Similarity search - Function
CTP synthase / CTP synthase, N-terminal / CTP synthase GATase domain / CTP synthase N-terminus / Glutamine amidotransferase class-I / Glutamine amidotransferase / Glutamine amidotransferase type 1 domain profile. / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / CYTIDINE-5'-TRIPHOSPHATE / CTP synthase / CTP synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsKollman, J.M. / Lynch, E.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
DepositionDec 8, 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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  • Biological unit as representative helical assembly
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)245,42912
Polymers241,7884
Non-polymers3,6418
Water0
1
A: CTP synthase
B: CTP synthase
C: CTP synthase
D: CTP synthase
hetero molecules
x 7


Theoretical massNumber of molelcules
Total (without water)1,718,00584
Polymers1,692,51528
Non-polymers25,49056
Water0
TypeNameSymmetry operationNumber
helical symmetry operation7
2


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
transform to helical frame1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 7 / Rise per n subunits: 81.591 Å / Rotation per n subunits: 48.563 °)

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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)
#2: Chemical
ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE / Cytidine triphosphate


Mass: 483.156 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H16N3O14P3
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: E. coli CTP synthase CC mutant filament / Type: COMPLEX
Details: Disulfide-crosslinked E. coli CTP synthase filaments assembled in non-reducing buffer, bound to CTP and ADP
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET22b
Buffer solutionpH: 8
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Filaments were assembled by dialysis in non-reducing buffer (50 mM Na-HEPES, pH 8.0). Products CTP (0.6 mM) and ADP (1.5 mM) were added to filaments.
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: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
4CTFFINDCTF correction
10SPIDERinitial Euler assignment
11SPIDERfinal Euler assignment
13SPIDER3D reconstruction
14hsearch_lorentz3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: 48.2 ° / Axial rise/subunit: 82.1 Å / Axial symmetry: C1
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31170 / Symmetry type: HELICAL

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