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- EMDB-35278: ecCTPS filament bound with CTP, NADH, DON -

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Basic information

Entry
Database: EMDB / ID: EMD-35278
TitleecCTPS filament bound with CTP, NADH, DON
Map data
Sample
  • Complex: ecCTPS filament with CTP,NADH,DON
    • Protein or peptide: CTP synthase
  • Ligand: ADENINE
  • Ligand: 5-OXO-L-NORLEUCINE
  • Ligand: CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsinhibitor / metabolic filament / CTP synthase / HYDROLASE
Function / homology
Function and homology information


CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / glutamine metabolic process / ATP binding / metal ion 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 speciesEscherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGuo CJ / Liu JL
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: mLife / Year: 2024
Title: Filamentation and inhibition of prokaryotic CTP synthase with ligands.
Authors: Chenjun Guo / Zixuan Wang / Ji-Long Liu /
Abstract: Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is ...Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.
History
DepositionFeb 7, 2023-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35278.png

Downloads & links

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Map

FileDownload / File: emd_35278.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.009183244 - 0.032624766
Average (Standard dev.)-0.00007616505 (±0.0012603642)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_35278_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35278_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35278_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ecCTPS filament with CTP,NADH,DON

EntireName: ecCTPS filament with CTP,NADH,DON
Components
  • Complex: ecCTPS filament with CTP,NADH,DON
    • Protein or peptide: CTP synthase
  • Ligand: ADENINE
  • Ligand: 5-OXO-L-NORLEUCINE
  • Ligand: CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ecCTPS filament with CTP,NADH,DON

SupramoleculeName: ecCTPS filament with CTP,NADH,DON / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: 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
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 60.317801 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MTTNYIFVTG GVVSSLGKGI AAASLAAILE ARGLNVTIMK LDPYINVDPG TMSPIQHGEV FVTEDGAETD LDLGHYERFI RTKMSRRNN FTTGRIYSDV LRKERRGDYL GATVQVIPHI TNAIKERVLE GGEGHDVVLV EIGGTVGDIE SLPFLEAIRQ M AVEIGREH ...String:
MTTNYIFVTG GVVSSLGKGI AAASLAAILE ARGLNVTIMK LDPYINVDPG TMSPIQHGEV FVTEDGAETD LDLGHYERFI RTKMSRRNN FTTGRIYSDV LRKERRGDYL GATVQVIPHI TNAIKERVLE GGEGHDVVLV EIGGTVGDIE SLPFLEAIRQ M AVEIGREH TLFMHLTLVP YMAASGEVKT KPTQHSVKEL LSIGIQPDIL ICRSDRAVPA NERAKIALFC NVPEKAVISL KD VDSIYKI PGLLKSQGLD DYICKRFSLN CPEANLSEWE QVIFEEANPV SEVTIGMVGK YIELPDAYKS VIEALKHGGL KNR VSVNIK LIDSQDVETR GVEILKGLDA ILVPGGFGYR GVEGMITTAR FARENNIPYL GICLGMQVAL IDYARHVANM ENAN STEFV PDCKYPVVAL ITEWRDENGN VEVRSEKSDL GGTMRLGAQQ CQLVDDSLVR QLYNAPTIVE RHRHRYEVNN MLLKQ IEDA GLRVAGRSGD DQLVEIIEVP NHPWFVACQF HPEFTSTPRD GHPLFAGFVK AASEFQKRQA

UniProtKB: CTP synthase

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Macromolecule #2: ADENINE

MacromoleculeName: ADENINE / type: ligand / ID: 2 / Number of copies: 4 / Formula: ADE
Molecular weightTheoretical: 135.127 Da
Chemical component information

ChemComp-ADE:
ADENINE

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Macromolecule #3: 5-OXO-L-NORLEUCINE

MacromoleculeName: 5-OXO-L-NORLEUCINE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ONL
Molecular weightTheoretical: 145.156 Da
Chemical component information

ChemComp-ONL:
5-OXO-L-NORLEUCINE

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Macromolecule #4: CYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: CYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 4 / Formula: CTP
Molecular weightTheoretical: 483.156 Da
Chemical component information

ChemComp-CTP:
CYTIDINE-5'-TRIPHOSPHATE

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1300114
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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