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- EMDB-61009: Focus refinement dmCTPS bound with dATP dUTP dGTP and DON -

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Basic information

Entry
Database: EMDB / ID: EMD-61009
TitleFocus refinement dmCTPS bound with dATP dUTP dGTP and DON
Map data
Sample
  • Complex: dmCTPS with dATP dUTP dGTP and DON
    • Protein or peptide: CTP synthase
  • Ligand: 6-DIAZENYL-5-OXO-L-NORLEUCINE
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYADENOSINE-5'-DIPHOSPHATE
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
  • 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
KeywordsCTPS / filament / inhibitor / substrates / intermediate / LIGASE
Function / homology
Function and homology information


Interconversion of nucleotide di- and triphosphates / larval lymph gland hemopoiesis / cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / ATP binding / identical protein binding ...Interconversion of nucleotide di- and triphosphates / larval lymph gland hemopoiesis / cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
CTP synthase / CTP synthase, N-terminal / CTP synthase GATase domain / CTP synthase N-terminus / Glutamine amidotransferase / Glutamine amidotransferase class-I / Glutamine amidotransferase type 1 domain profile. / 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: 2.79 Å
AuthorsGuo CJ / Liu JL
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: J Mol Biol / Year: 2024
Title: Structural Basis of Bifunctional CTP/dCTP Synthase.
Authors: Chen-Jun Guo / Zherong Zhang / Jia-Li Lu / Jiale Zhong / Yu-Fen Wu / Shu-Ying Guo / Ji-Long Liu /
Abstract: The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered ...The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully resolved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2'-deoxycytidine-5'-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to resolve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro, and provide a structural basis for these activities.
History
DepositionJul 31, 2024-
SupersessionNov 27, 2024ID: EMD-35102
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61009.png

Downloads & links

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Map

FileDownload / File: emd_61009.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 262.4 Å		1.03 Å/pix.
x 256 pix.
= 262.4 Å		1.03 Å/pix.
x 256 pix.
= 262.4 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.025 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.11901499 - 0.27551255
Average (Standard dev.)0.0006322611 (±0.0075304005)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61009_msk_1.map
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Additional map: #1

Fileemd_61009_additional_1.map
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Half map: #2

Fileemd_61009_half_map_1.map
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Half map: #1

Fileemd_61009_half_map_2.map
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Sample components

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Entire : dmCTPS with dATP dUTP dGTP and DON

EntireName: dmCTPS with dATP dUTP dGTP and DON
Components
  • Complex: dmCTPS with dATP dUTP dGTP and DON
    • Protein or peptide: CTP synthase
  • Ligand: 6-DIAZENYL-5-OXO-L-NORLEUCINE
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYADENOSINE-5'-DIPHOSPHATE
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
  • 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

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Supramolecule #1: dmCTPS with dATP dUTP dGTP and DON

SupramoleculeName: dmCTPS with dATP dUTP dGTP and DON / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Macromolecule #1: CTP synthase

MacromoleculeName: CTP synthase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: CTP synthase (glutamine hydrolysing)
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 62.443656 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 IQ

UniProtKB: CTP synthase

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Macromolecule #2: 6-DIAZENYL-5-OXO-L-NORLEUCINE

MacromoleculeName: 6-DIAZENYL-5-OXO-L-NORLEUCINE / type: ligand / ID: 2 / Number of copies: 1 / Formula: DON
Molecular weightTheoretical: 173.17 Da
Chemical component information

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

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: 2'-DEOXYADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: DAT
Molecular weightTheoretical: 411.202 Da
Chemical component information

ChemComp-DAT:
2'-DEOXYADENOSINE-5'-DIPHOSPHATE

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Macromolecule #5: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: DGT
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-DGT:
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

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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: 1 / 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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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 59.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 20.0 µm / Nominal defocus min: 5.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7dpt

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 832381
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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