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- EMDB-24512: Yeast CTP Synthase (URA8) Filament bound to ATP/UTP at low pH -

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Basic information

Entry
Database: EMDB / ID: EMD-24512
TitleYeast CTP Synthase (URA8) Filament bound to ATP/UTP at low pH
Map dataYeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH
Sample
  • Complex: Yeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH
    • Protein or peptide: CTP synthaseCTP synthetase
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: URIDINE 5'-TRIPHOSPHATE
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
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHansen JM / Lynch EM / Farrell DP / DiMaio F / Quispe J / Kollman JM
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateNov 24, 2021-
Current statusNov 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rl0
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24512.png

Downloads & links

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Map

FileDownload / File: emd_24512.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 2.9 / Movie #1: 2.9
Minimum - Maximum-14.687688 - 19.613884
Average (Standard dev.)1.6998881e-12 (±0.34325248)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-14.68819.6140.000

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Supplemental data

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Sample components

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Entire : Yeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH

EntireName: Yeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH
Components
  • Complex: Yeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH
    • Protein or peptide: CTP synthaseCTP synthetase
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: URIDINE 5'-TRIPHOSPHATE

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Supramolecule #1: Yeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH

SupramoleculeName: Yeast CTP Synthase (URA8) filament bound to ATP/UTP at low pH
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-CodonPlus (DE3)-RIL
Molecular weightExperimental: 256 kDa/nm

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Macromolecule #1: CTP synthase

MacromoleculeName: CTP synthase / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: CTP synthase (glutamine hydrolysing)
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 62.439168 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKYVVVSGGV ISGIGKGVLA SSTGMLLKTL GLKVTSIKID PYMNIDAGTM SPLEHGECFV LDDGGETDLD LGNYERYLGI TLSRDHNIT TGKIYSHVIS RERRGDYLGK TVQIVPHLTN AIQDWIQRVS KIPVDDTGLE PDVCIIELGG TVGDIESAPF V EALRQFQF ...String:
MKYVVVSGGV ISGIGKGVLA SSTGMLLKTL GLKVTSIKID PYMNIDAGTM SPLEHGECFV LDDGGETDLD LGNYERYLGI TLSRDHNIT TGKIYSHVIS RERRGDYLGK TVQIVPHLTN AIQDWIQRVS KIPVDDTGLE PDVCIIELGG TVGDIESAPF V EALRQFQF EVGRENFALI HVSLVPVIHG EQKTKPTQAA IKDLRSLGLI PDMIACRCSE ELNRSTIDKI AMFCHVGPEQ VV NVHDVNS TYHVPLLLLK QHMIDYLHSR LKLGEVPLTL EDKERGSQLL TNWENMTKNL DDSDDVVKIA LVGKYTNLKD SYL SVTKSL EHASMKCRRQ LEILWVEASN LEPETQEVDK NKFHDSWNKL SSADGILVPG GFGTRGIEGM ILAAKWARES GVPF LGVCL GLQVAAIEFA RNVIGRPNSS STEFLDETLL APEDQVVITM RLGLRPTIFQ PNSEWSNIRK LYGEVNEVHE RHRHR YEIN PKIVNDMESR GFIFVGKDET GQRCEIFELK GHPYYVGTQY HPEYTSKVLE PSRPFWGLVA AASGTLGEVI KDINL

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 24 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 12 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #4: URIDINE 5'-TRIPHOSPHATE

MacromoleculeName: URIDINE 5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 12 / Formula: UTP
Molecular weightTheoretical: 484.141 Da
Chemical component information

ChemComp-UTP:
URIDINE 5'-TRIPHOSPHATE / UTP*YM / Uridine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6
GridModel: C-flat / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 90.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: OTHER / Software - Name: RELION (ver. 3.1) / Details: FSCref0.5 (Phenix Density Modification) / Number images used: 40474

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Atomic model buiding 1

RefinementOverall B value: 32
Output model

PDB-7rl0:
Yeast CTP Synthase (URA8) Filament bound to ATP/UTP at low pH

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