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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/homologyCTP synthase / CTP synthase activity / CTP synthase GATase domain / CTP synthase, N-terminal / CTP synthase / CTP synthase N-terminus / 'de novo' CTP biosynthetic process / Glutamine amidotransferase / Glutamine amidotransferase type 1 domain profile. / CTP biosynthetic process ...CTP synthase / CTP synthase activity / CTP synthase GATase domain / CTP synthase, N-terminal / CTP synthase / 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
Function and homology information
Specimen sourceEscherichia coli / / bacteria /
MethodX-ray diffraction (2.7 Å resolution / Fourier synthesis) / X-ray crystallography
AuthorsBaldwin, E.P. / Endrizzi, J.A.
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 Report
SummaryFull reportAbout validation report
DateDeposition: Oct 7, 2016 / Release: Apr 26, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 26, 2017Structure modelrepositoryInitial release
1.1May 17, 2017Structure modelDatabase references
1.2Jun 14, 2017Structure modelDatabase referencescitation_citation.country / _citation.journal_volume / _citation.page_first / _citation.page_last
1.3Sep 20, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization
1.4Nov 1, 2017Structure modelAuthor supporting evidencepdbx_struct_assembly_auth_evidence

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)127,72120
Polyers125,1732
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
Polyers250,3454
Non-polymers5,09736
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
Buried area (Å2)20810
ΔGint (kcal/M)-395
Surface area (Å2)72530
MethodPISA
Unit cell
γ
α
β
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-MP 21 21 2
DetailsTetramer as determined by gel filtration and ultracentrifugation

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Components

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Protein/peptide , 1 types, 2 molecules AB

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


Mass: 62586.285 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli (strain K12) / Gene: pyrG, b2780, JW2751 / Plasmid name: pET28b / Production host: Escherichia coli / Strain (production host): BL21(DE3) / References: UniProt:P0A7E5, EC:6.3.4.2 (CTP synthase)

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Non-polymers , 7 types, 388 molecules

#2: Chemical ChemComp-GLN / GLUTAMINE


Mass: 146.144 Da / Num. of mol.: 2 / Formula: C5H10N2O3 / : Glutamine
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 8 / Formula: SO4 / : Sulfate
#4: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 2 / Formula: C6H14O2 / : 2-Methyl-2,4-pentanediol
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Formula: Mg / : Magnesium
#6: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 2 / Formula: C9H16N3O14P3 / : Cytidine triphosphate
#7: Chemical ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL


Mass: 118.174 Da / Num. of mol.: 2 / Formula: C6H14O2 / : 2-Methyl-2,4-pentanediol
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 370 / Formula: H2O / : Water

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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 / Density percent sol: 73 / Description: Rectangular Prisms
Crystal growTemp: 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 kelvins
SourceSource: SYNCHROTRON / Type: SSRL BEAMLINE BL1-5 / Synchrotron site: SSRL / Beamline: BL1-5 / Wavelength: 0.97961
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Collection date: Mar 26, 2005
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97961 Å / Relative weight: 1
ReflectionB iso Wilson estimate: 40 Å2 / D resolution high: 2.7 Å / D resolution low: 30.02 Å / Number obs: 62343 / Rmerge I obs: 0.092 / Rsym value: 0.072 / NetI over sigmaI: 11.2 / Redundancy: 3.4 / Percent possible obs: 98.3
Reflection shellRmerge I obs: 0.31 / Highest resolution: 2.7 Å / Lowest resolution: 2.82 Å / MeanI over sigI obs: 3.1 / Redundancy: 3.2 / Percent possible all: 99

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Processing

Software
NameVersionClassification
REFMAC5.6.0117refinement
HKL-2000data reduction
HKL-2000data scaling
REFMAC5.6.0117phasing
RefineMethod to determine structure: FOURIER SYNTHESIS
Starting model: 2AD5
Correlation coeff Fo to Fc: 0.959 / Correlation coeff Fo to Fc free: 0.929 / R Free selection details: RANDOM / Cross valid method: THROUGHOUT / Overall ESU R: 0.265 / Overall ESU R Free: 0.222 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Solvent computationSolvent ion probe radii: 0.8 Å / Solvent shrinkage radii: 0.8 Å / Solvent vdw probe radii: 1.2 Å / Solvent model details: BABINET MODEL WITH MASK
Displacement parametersB iso mean: 46.541 Å2 / Aniso B11: -1.82 Å2 / Aniso B12: 0 Å2 / Aniso B13: 0 Å2 / Aniso B22: 2.83 Å2 / Aniso B23: 0 Å2 / Aniso B33: -1.01 Å2
Least-squares processR factor R free: 0.20764 / R factor R work: 0.15749 / R factor obs: 0.16005 / Highest resolution: 2.7 Å / Lowest resolution: 30.02 Å / Number reflection R free: 3159 / Number reflection obs: 59184 / Percent reflection R free: 5.1 / Percent reflection obs: 98.22
Refine hist #1Highest resolution: 2.7 Å / Lowest resolution: 30.02 Å
Number of atoms included #1Protein: 8305 / Nucleic acid: 0 / Ligand: 152 / Solvent: 370 / Total: 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_deg2.4501.98111663
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.4825.0001063
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.03924.133392
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.83115.0001468
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.86115.00064
X-RAY DIFFRACTIONr_chiral_restr0.1640.2001331
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
Refine LS shellHighest resolution: 2.7 Å / R factor R free: 0.328 / R factor R work: 0.235 / Lowest resolution: 2.77 Å / Number reflection R free: 233 / Number reflection R work: 4315 / Total number of bins used: 20 / Percent reflection obs: 98.98

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