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- PDB-7rkh: Yeast CTP Synthase (URA8) tetramer bound to ATP/UTP at neutral pH -

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Basic information

Entry
Database: PDB / ID: 7rkh
TitleYeast CTP Synthase (URA8) tetramer bound to ATP/UTP at neutral pH
ComponentsCTP synthaseCTP synthetase
KeywordsPROTEIN FIBRIL / glutaminase / amido-ligase / nucleotide metabolism
Function / homology
Function and homology information


CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / glutamine metabolic process / ATP binding
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'-TRIPHOSPHATE / URIDINE 5'-TRIPHOSPHATE / CTP synthase
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
Model detailscryo-EM D2 reconstruction
AuthorsHansen, J.M. / Lynch, E.M. / Farrell, D.P. / DiMaio, F. / Quispe, J. / Kollman, J.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM118396 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM007270 United States
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 22, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 24, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)253,91620
Polymers249,7574
Non-polymers4,16016
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, SDS gel and cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17730 Å2
ΔGint-69 kcal/mol
Surface area81680 Å2

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Components

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


Mass: 62439.168 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: 6XHIS c-ter
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS3439, SCNYR20_0009027000 / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-CodonPlus (DE3)-RIL
References: UniProt: A0A6A5PYW3, CTP synthase (glutamine hydrolysing)
#2: Chemical
ChemComp-UTP / URIDINE 5'-TRIPHOSPHATE / Uridine triphosphate


Mass: 484.141 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Comment: UTP*YM
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
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: Yeast CTP Synthase (URA8) tetramer bound to ATP/UTP at neutral pH
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 256 kDa/nm / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-CodonPlus (DE3)-RIL
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 5500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 90 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
10RELION3.1final Euler assignment
12RELION3D reconstruction
13ISOLDE1.1.0model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.8 Å / Resolution method: OTHER / Num. of particles: 76963 / Details: FSCref0.5 (Phenix Density modification) / Symmetry type: POINT

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