|Entry||Database: PDB / ID: 7rkh|
|Title||Yeast CTP Synthase (URA8) tetramer bound to ATP/UTP at neutral pH|
|Components||CTP synthaseCTP synthetase|
|Keywords||PROTEIN 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 / 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 species||Saccharomyces cerevisiae (baker's yeast)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å|
|Model details||cryo-EM D2 reconstruction|
|Authors||Hansen, J.M. / Lynch, E.M. / Farrell, D.P. / DiMaio, F. / Quispe, J. / Kollman, J.M.|
|Funding support|| United States, 2items |
|Citation||Journal: 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 polymerization and investigate 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.
|Structure viewer||Molecule: |
Downloads & links
D: CTP synthase
A: CTP synthase
B: CTP synthase
C: CTP synthase
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 (baker'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)
Mass: 484.141 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Comment: UTP*YM
Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
|Has ligand of interest||N|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Yeast CTP Synthase (URA8) tetramer bound to ATP/UTP at neutral pH|
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
|Molecular weight||Value: 256 kDa/nm / Experimental value: YES|
|Source (natural)||Organism: Saccharomyces cerevisiae (baker's yeast)|
|Source (recombinant)||Organism: Escherichia coli (E. coli) / Strain: BL21-CodonPlus (DE3)-RIL|
|Buffer solution||pH: 7.4|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 5500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Electron dose: 90 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|EM imaging optics||Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV|
|Image scans||Movie frames/image: 50|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: D2 (2x2 fold dihedral)|
|3D reconstruction||Resolution: 2.8 Å / Resolution method: OTHER / Num. of particles: 76963 / Details: FSCref0.5 (Phenix Density modification) / Symmetry type: POINT|
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