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- PDB-8rht: Crystal Structure of Trypanosoma brucei DHFR in complex with the ... -

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Basic information

Entry
Database: PDB / ID: 8rht
TitleCrystal Structure of Trypanosoma brucei DHFR in complex with the cofactor and inhibitor P25
ComponentsBifunctional dihydrofolate reductase-thymidylate synthase
KeywordsOXIDOREDUCTASE / Trypanosoma brucei / DHFR / Dihydrofolate Reductase / cofactor / NADPH / inhibitor
Function / homology
Function and homology information


thymidylate synthase / thymidylate synthase activity / dTMP biosynthetic process / dTTP biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / methylation / mitochondrion / cytosol
Similarity search - Function
Bifunctional dihydrofolate reductase/thymidylate synthase / Thymidylate synthase, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase/dCMP hydroxymethylase superfamily / Thymidylate synthase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. ...Bifunctional dihydrofolate reductase/thymidylate synthase / Thymidylate synthase, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase/dCMP hydroxymethylase superfamily / Thymidylate synthase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily
Similarity search - Domain/homology
: / Chem-NDP / Bifunctional dihydrofolate reductase-thymidylate synthase
Similarity search - Component
Biological speciesTrypanosoma brucei brucei (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsPozzi, C. / Mangani, S. / Landi, G.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Union (EU)CA21111European Union
CitationJournal: Acs Infect Dis. / Year: 2024
Title: Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti- Trypanosoma brucei Agents.
Authors: Francesconi, V. / Rizzo, M. / Pozzi, C. / Tagliazucchi, L. / Konchie Simo, C.U. / Saporito, G. / Landi, G. / Mangani, S. / Carbone, A. / Schenone, S. / Santarem, N. / Tavares, J. / Cordeiro- ...Authors: Francesconi, V. / Rizzo, M. / Pozzi, C. / Tagliazucchi, L. / Konchie Simo, C.U. / Saporito, G. / Landi, G. / Mangani, S. / Carbone, A. / Schenone, S. / Santarem, N. / Tavares, J. / Cordeiro-da-Silva, A. / Costi, M.P. / Tonelli, M.
History
DepositionDec 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,5783
Polymers26,5901
Non-polymers9892
Water41423
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1260 Å2
ΔGint-4 kcal/mol
Surface area10190 Å2
MethodPISA
Unit cell
Length a, b, c (Å)41.920, 60.928, 68.882
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Bifunctional dihydrofolate reductase-thymidylate synthase / DHFR-TS


Mass: 26589.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): ArcticExpress (DE3)
References: UniProt: Q27783, dihydrofolate reductase, thymidylate synthase
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#3: Chemical ChemComp-A1H0S / 4,4,7,8-tetramethyl-10~{H}-[1,3,5]triazino[1,2-a]benzimidazol-2-amine / Mecillinam, bound form


Mass: 243.308 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H17N5 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 1.6 Å3/Da / Density % sol: 23.28 %
Crystal growTemperature: 293 K / Method: microbatch / pH: 6.5
Details: 35% w/v PEG4000, 50 mM ammonium sulfate, 50 mM bis-tris, pH 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.99989 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 19, 2023
RadiationMonochromator: Double Crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.99989 Å / Relative weight: 1
ReflectionResolution: 2.9→60.93 Å / Num. obs: 4243 / % possible obs: 100 % / Observed criterion σ(I): 2 / Redundancy: 6.3 % / Biso Wilson estimate: 25.2 Å2 / CC1/2: 0.996 / Rmerge(I) obs: 0.124 / Rpim(I) all: 0.077 / Rrim(I) all: 0.147 / Net I/σ(I): 8.6
Reflection shellResolution: 2.9→3.08 Å / Redundancy: 6.6 % / Rmerge(I) obs: 0.964 / Mean I/σ(I) obs: 2.1 / Num. unique obs: 669 / CC1/2: 0.92 / Rpim(I) all: 0.592 / Rrim(I) all: 1.134 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0419refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.9→45.64 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.894 / SU B: 30.028 / SU ML: 0.545 / Cross valid method: THROUGHOUT / ESU R Free: 0.57 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.29797 186 4.4 %RANDOM
Rwork0.18778 ---
obs0.19274 4027 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 75.303 Å2
Baniso -1Baniso -2Baniso -3
1-0.08 Å20 Å20 Å2
2--0.01 Å2-0 Å2
3----0.09 Å2
Refine analyzeLuzzati coordinate error obs: 0.4 Å
Refinement stepCycle: 1 / Resolution: 2.9→45.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1612 0 66 23 1701
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.0121716
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.5811.8522349
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.0975216
X-RAY DIFFRACTIONr_dihedral_angle_2_deg6.003519
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.55410257
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.1190.2271
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.021283
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_it5.7877.75867
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it9.54313.9191082
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it6.0657.684849
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined15.69493.242672
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: 2.9→2.975 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.457 18 -
Rwork0.335 296 -
obs--100 %

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