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- PDB-7rmk: Yeast CTP Synthase (Ura7) Bundle bound to substrates at low pH -

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Basic information

Entry
Database: PDB / ID: 7rmk
TitleYeast CTP Synthase (Ura7) Bundle bound to substrates at low pH
ComponentsCTP synthase
KeywordsPROTEIN FIBRIL / glutaminase and amino-ligase
Function / homology
Function and homology information


cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / glutamine metabolic process / ATP binding / identical protein binding / cytosol
Similarity search - Function
CTP synthase / CTP synthase, N-terminal / CTP synthase GATase domain / CTP synthase N-terminus / Glutamine amidotransferase / Glutamine amidotransferase class-I / Glutamine amidotransferase type 1 domain profile. / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / URIDINE 5'-TRIPHOSPHATE / CTP synthase
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å
Model detailscryo-EM D2 reconstruction
AuthorsHansen, J.M. / Lynch, E.M. / Farrell, D.P. / DiMaio, F. / Quispe, J. / Kollman, J.M.
Funding support2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM118396
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM007270
CitationJournal: Elife / Year: 2021
Title: Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation.
Authors: Jesse M Hansen / Avital Horowitz / Eric M Lynch / Daniel P Farrell / Joel Quispe / Frank DiMaio / Justin M Kollman /
Abstract: Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in ...Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.
History
DepositionJul 27, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 24, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
R: CTP synthase
K: CTP synthase
L: CTP synthase
M: CTP synthase
Y: CTP synthase
I: CTP synthase
P: CTP synthase
U: CTP synthase
D: CTP synthase
A: CTP synthase
B: CTP synthase
C: CTP synthase
Z: CTP synthase
J: CTP synthase
Q: CTP synthase
V: CTP synthase
H: CTP synthase
E: CTP synthase
F: CTP synthase
G: CTP synthase
a: CTP synthase
N: CTP synthase
S: CTP synthase
W: CTP synthase
b: CTP synthase
O: CTP synthase
T: CTP synthase
X: CTP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,760,39584
Polymers1,732,63828
Non-polymers27,75756
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
CTP synthase / UTP--ammonia ligase


Mass: 61879.922 Da / Num. of mol.: 28
Source method: isolated from a genetically manipulated source
Details: 6XHIS c-ter
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS169, PACBIOSEQ_LOCUS176, PACBIOSEQ_LOCUS177, PACBIOSEQ_LOCUS179
Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-CodonPlus (DE3)-RIL
References: UniProt: A0A7I9CFN1, CTP synthase (glutamine hydrolysing)
#2: Chemical...
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 28 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#3: Chemical...
ChemComp-UTP / URIDINE 5'-TRIPHOSPHATE


Mass: 484.141 Da / Num. of mol.: 28 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Comment: UTP*YM
Has ligand of interestN

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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: CTP Synthase Bundle assembled with substrates / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 90 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24059 / Symmetry type: POINT

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