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- PDB-5tkv: X-RAY CRYSTAL STRUCTURE OF THE "CLOSED" CONFORMATION OF CTP-INHIB... -

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Basic information

Entry
Database: PDB / ID: 5tkv
TitleX-RAY CRYSTAL STRUCTURE OF THE "CLOSED" CONFORMATION OF CTP-INHIBITED E. COLI CYTIDINE TRIPHOSPHATE (CTP) SYNTHETASE
ComponentsCTP synthaseCTP synthetase
KeywordsLYASE / PYRIMIDINE BIOSYNTHESIS / ENZYME REGULATION VIA POLYMERIZATION / FEEDBACK INHIBITION
Function / homology
Function and homology information


cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / glutaminase activity / CTP biosynthetic process / glutamine metabolic process / protein homotetramerization / magnesium ion binding ...cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / glutaminase activity / CTP biosynthetic process / glutamine metabolic process / protein homotetramerization / magnesium ion binding / protein-containing complex / ATP binding / identical protein binding / cytosol
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 (GATase) domain / Class I glutamine amidotransferase-like / P-loop containing nucleotide triphosphate hydrolases ...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 (GATase) domain / Class I glutamine amidotransferase-like / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
CYTIDINE-5'-TRIPHOSPHATE / GLUTAMINE / CTP synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.7 Å
AuthorsBaldwin, E.P. / Endrizzi, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1-GM63109 United States
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.
History
DepositionOct 7, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2017Provider: repository / Type: Initial release
Revision 1.1May 17, 2017Group: Database references
Revision 1.2Jun 14, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_volume ..._citation.country / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.5Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CTP synthase
B: CTP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,72120
Polymers125,1732
Non-polymers2,54818
Water6,666370
1
A: CTP synthase
B: CTP synthase
hetero molecules

A: CTP synthase
B: CTP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,44240
Polymers250,3454
Non-polymers5,09736
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
Buried area20810 Å2
ΔGint-395 kcal/mol
Surface area72530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)159.159, 110.675, 129.487
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212
SymmetryPoint symmetry: (Schoenflies symbol: D2 (2x2 fold dihedral))
DetailsTetramer as determined by gel filtration and ultracentrifugation

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein CTP synthase / CTP synthetase / Cytidine 5'-triphosphate synthase / Cytidine triphosphate synthetase / CTPS / UTP--ammonia ligase


Mass: 62586.285 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: pyrG, b2780, JW2751 / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: P0A7E5, CTP synthase (glutamine hydrolysing)

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Non-polymers , 7 types, 388 molecules

#2: Chemical ChemComp-GLN / GLUTAMINE / Glutamine


Type: L-peptide linking / Mass: 146.144 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H10N2O3
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL / 2-Methyl-2,4-pentanediol


Mass: 118.174 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE / Cytidine triphosphate


Mass: 483.156 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H16N3O14P3
#7: Chemical ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL / 2-Methyl-2,4-pentanediol


Mass: 118.174 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 370 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.6 Å3/Da / Density % sol: 73 % / Description: Rectangular Prisms
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 0.8 M Ammonium Sulfate 10 mM CTP 10 mM Glutamine 10 mM Magnesium Sulfate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL1-5 / Wavelength: 0.97961 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Mar 26, 2005
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97961 Å / Relative weight: 1
ReflectionResolution: 2.7→30.02 Å / Num. obs: 62343 / % possible obs: 98.3 % / Redundancy: 3.4 % / Biso Wilson estimate: 40 Å2 / Rmerge(I) obs: 0.092 / Rsym value: 0.072 / Net I/σ(I): 11.2
Reflection shellResolution: 2.7→2.82 Å / Redundancy: 3.2 % / Rmerge(I) obs: 0.31 / Mean I/σ(I) obs: 3.1 / % possible all: 99

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Processing

Software
NameVersionClassification
REFMAC5.6.0117refinement
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.6.0117phasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 2AD5

2ad5


Resolution: 2.7→30.02 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.929 / Cross valid method: THROUGHOUT / ESU R: 0.265 / ESU R Free: 0.222 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.20764 3159 5.1 %RANDOM
Rwork0.15749 ---
obs0.16005 59184 98.22 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 46.541 Å2
Baniso -1Baniso -2Baniso -3
1--1.82 Å20 Å20 Å2
2--2.83 Å20 Å2
3----1.01 Å2
Refinement stepCycle: 1 / Resolution: 2.7→30.02 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8305 0 152 370 8827
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0240.0198595
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg2.451.98111663
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.48251063
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.03924.133392
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.831151468
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.8611564
X-RAY DIFFRACTIONr_chiral_restr0.1640.21331
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0216430
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.328 233 -
Rwork0.235 4315 -
obs--98.98 %

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