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- EMDB-30810: Structural basis for ligand binding modes of CTP synthase -

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Basic information

Entry
Database: EMDB / ID: EMD-30810
TitleStructural basis for ligand binding modes of CTP synthase
Map data
Sample
  • Organelle or cellular component: Drosophila substrate-bound CTP synthase
    • Protein or peptide: CTP synthaseCTP synthetase
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: 6-DIAZENYL-5-OXO-L-NORLEUCINE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: [[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(2-oxidanyl-4-phosphonooxy-pyrimidin-1-yl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate
  • Ligand: water
Function / homology
Function and homology information


Interconversion of nucleotide di- and triphosphates / larval lymph gland hemopoiesis / CTP synthase activity / cytoophidium / CTP synthase (glutamine hydrolysing) / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / glutamine metabolic process / ATP binding ...Interconversion of nucleotide di- and triphosphates / larval lymph gland hemopoiesis / CTP synthase activity / cytoophidium / CTP synthase (glutamine hydrolysing) / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / glutamine metabolic process / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
CTP synthase N-terminus / CTP synthase GATase domain / CTP synthase, N-terminal / CTP synthase / Glutamine amidotransferase type 1 domain profile. / Glutamine amidotransferase class-I / Glutamine amidotransferase / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsLiu JL / Zhou X / Guo CJ / Chang CC
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structural basis for ligand binding modes of CTP synthase.
Authors: Xian Zhou / Chen-Jun Guo / Chia-Chun Chang / Jiale Zhong / Huan-Huan Hu / Guang-Ming Lu / Ji-Long Liu /
Abstract: Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is ...Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.
History
DepositionDec 21, 2020-
Header (metadata) releaseSep 15, 2021-
Map releaseSep 15, 2021-
UpdateSep 15, 2021-
Current statusSep 15, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
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  • Surface view with fitted model
  • Atomic models: PDB-7dpt
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7dpt
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_30810.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 250 pix.
= 265. Å
1.06 Å/pix.
x 250 pix.
= 265. Å
1.06 Å/pix.
x 250 pix.
= 265. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.40901485 - 0.8677972
Average (Standard dev.)7.2480914e-05 (±0.016204491)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 265.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z265.000265.000265.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ330330330
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.4090.8680.000

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Supplemental data

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Mask #1

Fileemd_30810_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_30810_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_30810_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Drosophila substrate-bound CTP synthase

EntireName: Drosophila substrate-bound CTP synthase
Components
  • Organelle or cellular component: Drosophila substrate-bound CTP synthase
    • Protein or peptide: CTP synthaseCTP synthetase
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: 6-DIAZENYL-5-OXO-L-NORLEUCINE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: [[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(2-oxidanyl-4-phosphonooxy-pyrimidin-1-yl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate
  • Ligand: water

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Supramolecule #1: Drosophila substrate-bound CTP synthase

SupramoleculeName: Drosophila substrate-bound CTP synthase / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Macromolecule #1: CTP synthase

MacromoleculeName: CTP synthase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: CTP synthase (glutamine hydrolysing)
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 69.539453 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MKYILVTGGV ISGVGKGVIA SSFGTLLKSC GLDVTSIKID PYINIDAGTF SPYEHGEVYV LDDGAEVDLD LGNYERFLDV TLHRDNNIT TGKIYKLVIE KERTGEYLGK TVQVVPHITD AIQEWVERVA QTPVQGSSKP QVCIVELGGT IGDIEGMPFV E AFRQFQFR ...String:
MKYILVTGGV ISGVGKGVIA SSFGTLLKSC GLDVTSIKID PYINIDAGTF SPYEHGEVYV LDDGAEVDLD LGNYERFLDV TLHRDNNIT TGKIYKLVIE KERTGEYLGK TVQVVPHITD AIQEWVERVA QTPVQGSSKP QVCIVELGGT IGDIEGMPFV E AFRQFQFR VKRENFCLAH VSLVPLPKAT GEPKTKPTQS SVRELRGCGL SPDLIVCRSE KPIGLEVKEK ISNFCHVGPD QV ICIHDLN SIYHVPLLME QNGVIEYLNE RLQLNIDMSK RTKCLQQWRD LARRTETVRR EVCIAVVGKY TKFTDSYASV VKA LQHAAL AVNRKLELVF IESCLLEEET LHSEPSKYHK EWQKLCDSHG ILVPGGFGSR GMEGKIRACQ WARENQKPLL GICL GLQAA VIEFARNKLG LKDANTTEID PNTANALVID MPEHHTGQLG GTMRLGKRIT VFSDGPSVIR QLYGNPKSVQ ERHRH RYEV NPKYVHLLEE QGMRFVGTDV DKTRMEIIEL SGHPYFVATQ YHPEYLSRPL KPSPPFLGLI LASVDRLNQY IQRGCR LSP RQLSDASSDE EDSVVGLAGA TKSLSSLKIP ITPTNGISKS CNGSISTSDS EGACGGVDPT NGHK

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Macromolecule #2: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #3: 6-DIAZENYL-5-OXO-L-NORLEUCINE

MacromoleculeName: 6-DIAZENYL-5-OXO-L-NORLEUCINE / type: ligand / ID: 3 / Number of copies: 4 / Formula: DON
Molecular weightTheoretical: 173.17 Da
Chemical component information

ChemComp-DON:
6-DIAZENYL-5-OXO-L-NORLEUCINE

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: [[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(2-oxidanyl-4-pho...

MacromoleculeName: [[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(2-oxidanyl-4-phosphonooxy-pyrimidin-1-yl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate
type: ligand / ID: 6 / Number of copies: 4 / Formula: 5ZL
Molecular weightTheoretical: 565.129 Da
Chemical component information

ChemComp-5ZL:
[[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(2-oxidanyl-4-phosphonooxy-pyrimidin-1-yl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 96 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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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: DARK FIELD
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 107556
FSC plot (resolution estimation)

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