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- EMDB-8504: CryoEM structure of the CTP synthase filament at 4.6 Angstrom res... -

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Basic information

Entry
Database: EMDB / ID: 8504
TitleCryoEM structure of the CTP synthase filament at 4.6 Angstrom resolution
Map dataCTP synthase filament
SampleCTP synthase filament
  • CTP synthaseCTP synthetase
  • (ligand) x 2
Function/homologyCTP synthase / CTP synthase / CTP synthase GATase domain / CTP synthase activity / CTP synthase, N-terminal / CTP synthase N-terminus / 'de novo' CTP biosynthetic process / Glutamine amidotransferase / Glutamine amidotransferase type 1 domain profile. / CTP biosynthetic process ...CTP synthase / CTP synthase / CTP synthase GATase domain / CTP synthase activity / CTP synthase, N-terminal / CTP synthase N-terminus / 'de novo' CTP biosynthetic process / Glutamine amidotransferase / Glutamine amidotransferase type 1 domain profile. / CTP biosynthetic process / Glutamine amidotransferase class-I / glutamine metabolic process / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase / ATP binding / identical protein binding / metal ion binding / cytosol / CTP synthase / CTP synthase
Function and homology information
SourceEscherichia coli / / bacteria /
MethodCryo EM / helical reconstruction / 4.6 Å resolution
AuthorsKollman JM / Lynch EM
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.
Validation ReportPDB-ID: 5u3c

SummaryFull reportAbout validation report
DateDeposition: Dec 1, 2016 / Header (metadata) release: Feb 8, 2017 / Map release: Apr 26, 2017 / Last update: Jun 21, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF CHIMERA
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  • Surface view with fitted model
  • Atomic models: : PDB-5u3c
  • Surface level: 2
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_8504.map.gz (map file in CCP4 format, 131073 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.22 Å/pix.
= 390.4 Å
320 pix
1.22 Å/pix.
= 390.4 Å
320 pix
1.22 Å/pix.
= 390.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.22 Å
Density
Contour Level:2 (by author), 2 (movie #1):
Minimum - Maximum-3.8410912 - 7.7650504
Average (Standard dev.)6.474641E-9 (0.40380588)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin-160-160-160
Limit159159159
Spacing320320320
CellA=B=C: 390.40002 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.221.221.22
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z390.400390.400390.400
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-160-160-160
NC/NR/NS320320320
D min/max/mean-3.8417.7650.000

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Supplemental data

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Sample components

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Entire CTP synthase filament

EntireName: CTP synthase filament
Details: CTP synthase was incubated in the presence of substrates, and filaments form as the product CTP builds up in the reaction.
Number of components: 4

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Component #1: protein, CTP synthase filament

ProteinName: CTP synthase filament
Details: CTP synthase was incubated in the presence of substrates, and filaments form as the product CTP builds up in the reaction.
Recombinant expression: No
SourceSpecies: Escherichia coli / / bacteria /
Source (engineered)Expression System: Escherichia coli / / bacteria / / Vector: pET22

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Component #2: protein, CTP synthase

ProteinName: CTP synthaseCTP synthetase / Recombinant expression: No
MassTheoretical: 60.44698 kDa
Source (engineered)Expression System: Escherichia coli / / bacteria /

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Component #3: ligand, CYTIDINE-5'-TRIPHOSPHATE

LigandName: CYTIDINE-5'-TRIPHOSPHATE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.483156 kDa

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Component #4: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: Cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 81.6 Å / Delta phi: 48.5 deg.
Sample solutionSpecimen conc.: 0.85 mg/ml / pH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 34 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: SPIDER / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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