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- PDB-2c2s: Human Dihydrofolate Reductase Complexed With NADPH and 2,4-Diamin... -

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Basic information

Entry
Database: PDB / ID: 2c2s
TitleHuman Dihydrofolate Reductase Complexed With NADPH and 2,4-Diamino-5-(1-o-carboranylmethyl)-6-methylpyrimidine, A novel boron containing, nonclassical Antifolate
ComponentsDIHYDROFOLATE REDUCTASE
KeywordsOXIDOREDUCTASE / NONCLASSICAL ANTIFOLATES / LIPOPHILIC ANTIFOLATES / NADP / ONE- CARBON METABOLISM / REDUCTASE
Function / homology
Function and homology information


regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / Metabolism of folate and pterines / tetrahydrofolate metabolic process / response to methotrexate / sequence-specific mRNA binding / axon regeneration / folic acid binding / dihydrofolate metabolic process / G1/S-Specific Transcription ...regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / Metabolism of folate and pterines / tetrahydrofolate metabolic process / response to methotrexate / sequence-specific mRNA binding / axon regeneration / folic acid binding / dihydrofolate metabolic process / G1/S-Specific Transcription / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / NADPH binding / tetrahydrofolate biosynthetic process / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / one-carbon metabolic process / positive regulation of nitric-oxide synthase activity / mRNA regulatory element binding translation repressor activity / NADP binding / negative regulation of translation / mRNA binding / mitochondrion / cytosol
Similarity search - Function
Dihydrofolate reductase / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-34B / Chem-NDP / Dihydrofolate reductase
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsLeung, A.K.W. / Reynolds, R.C. / Riordan, J.M. / Borhani, D.W.
CitationJournal: J.Med.Chem. / Year: 2007
Title: Novel Boron-Containing, Nonclassical Antifolates: Synthesis and Preliminary Biological and Structural Evaluation.
Authors: Reynolds, R.C. / Campbell, S.R. / Fairchild, R.G. / Kisliuk, R.L. / Micca, P.L. / Queener, S.F. / Riordan, J.M. / Sedwick, W.D. / Waud, W.R. / Leung, A.K.W. / Dixon, R.W. / Suling, W.J. / Borhani, D.W.
History
DepositionSep 29, 2005Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2007Provider: repository / Type: Initial release
Revision 1.1May 7, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Mar 6, 2019Group: Data collection / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Revision 1.4May 8, 2019Group: Data collection / Experimental preparation
Category: database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow
Item: _exptl_crystal_grow.method
Revision 1.5May 1, 2024Group: Data collection / Database references ...Data collection / Database references / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf
Remark 700 SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN ... SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN THE SHEET RECORDS BELOW, TWO SHEETS ARE DEFINED.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DIHYDROFOLATE REDUCTASE
B: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,8437
Polymers42,6992
Non-polymers2,1445
Water10,016556
1
A: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,3753
Polymers21,3501
Non-polymers1,0262
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
2
B: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,4674
Polymers21,3501
Non-polymers1,1183
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)87.671, 93.864, 95.782
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11B-2280-

HOH

Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (-0.064193, 0.996943, -0.04455), (-0.995562, -0.067055, -0.066028), (-0.068813, 0.040114, 0.996823)
Vector: -2.4257, 47.20213, 22.86397)

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Components

#1: Protein DIHYDROFOLATE REDUCTASE


Mass: 21349.525 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PDFR / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00374, dihydrofolate reductase
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#3: Chemical ChemComp-34B / 2,4-DIAMINO-5-(1-O-CARBORANYLMETHYL)-6-METHYLPYRIMIDINE


Mass: 280.381 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H20B10N4
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 556 / 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: 2.3 Å3/Da / Density % sol: 46.3 %
Description: INTENSITIES WERE CONVERTED TO STRUCTURE FACTORS USING CCP4 PROGRAM TRUNCATE.
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: THE TERNARY COMPLEX OF DHFR WITH NADPH AND 34B WAS FORMED BY MIXING HUMAN DHFR (20 MG/ML IN 25 MM KPO4 (PH 7.0), 0.1 MM EDTA, AND 3 MM NAN3) WITH 60 MM NADPH, FOLLOWED 15 MINUTES LATER BY 60 ...Details: THE TERNARY COMPLEX OF DHFR WITH NADPH AND 34B WAS FORMED BY MIXING HUMAN DHFR (20 MG/ML IN 25 MM KPO4 (PH 7.0), 0.1 MM EDTA, AND 3 MM NAN3) WITH 60 MM NADPH, FOLLOWED 15 MINUTES LATER BY 60 MM OF INHIBITOR IN DMSO (FINAL CONCENTRATIONS OF 2 MM NADPH AND 2 MM INHIBITOR). THIS COMPLEX SOLUTION WAS MIXED WITH AN EQUAL VOLUME OF PRECIPITANT CONTAINING 24-33% PEG 4000, 0.2 M LI2SO4, 0.1 M TRIS CL (PH 7.9-8.4), 5% GLYCEROL, AND EQUILIBRATED WITH THE PRECIPITANT BY HANGING DROP VAPOR DIFFUSION AT 277 K. THE CRYSTAL GREW IN ABOUT 3 WEEKS. THE CRYSTAL WAS FLASH-COOLED DIRECTLY IN LIQUID NITROGEN AFTER HARVESTING INTO MOTHER LIQUOR CONTAINING 10% GLYCEROL.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-1 / Wavelength: 0.783
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Mar 2, 1999 / Details: MIRROR
RadiationMonochromator: SI (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.783 Å / Relative weight: 1
ReflectionResolution: 1.4→35 Å / Num. obs: 65300 / % possible obs: 84 % / Observed criterion σ(I): 0 / Redundancy: 3 % / Biso Wilson estimate: 16.1 Å2 / Rmerge(I) obs: 0.05 / Net I/σ(I): 11.2
Reflection shellResolution: 1.4→1.44 Å / Redundancy: 1.3 % / Rmerge(I) obs: 0.46 / Mean I/σ(I) obs: 1.5 / % possible all: 43.1

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Processing

Software
NameVersionClassification
REFMAC5.2.0019refinement
MOSFLMdata reduction
SCALAdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: UNPUBLISHED HUMAN DHFR STRUCTURE

Resolution: 1.4→20 Å / Cor.coef. Fo:Fc: 0.972 / Cor.coef. Fo:Fc free: 0.958 / SU B: 2.682 / SU ML: 0.048 / Cross valid method: THROUGHOUT / ESU R: 0.086 / ESU R Free: 0.074 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.198 3260 5 %RANDOM
Rwork0.155 ---
obs0.157 61984 83.9 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 19.68 Å2
Baniso -1Baniso -2Baniso -3
1--0.16 Å20 Å20 Å2
2--0.18 Å20 Å2
3----0.02 Å2
Refinement stepCycle: LAST / Resolution: 1.4→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2962 0 146 556 3664
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0223344
X-RAY DIFFRACTIONr_bond_other_d0.0010.022324
X-RAY DIFFRACTIONr_angle_refined_deg1.5532.0174726
X-RAY DIFFRACTIONr_angle_other_deg1.2132.9795769
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0225374
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.12325.278144
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.6515595
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.3021513
X-RAY DIFFRACTIONr_chiral_restr0.0940.2493
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.023465
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02580
X-RAY DIFFRACTIONr_nbd_refined0.2030.2512
X-RAY DIFFRACTIONr_nbd_other0.1920.22256
X-RAY DIFFRACTIONr_nbtor_refined0.1770.21486
X-RAY DIFFRACTIONr_nbtor_other0.0850.21688
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1320.2331
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1740.210
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2020.264
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1470.247
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: 1.4→1.44 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.342 122 -
Rwork0.213 2295 -
obs--42.6 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.00980.32-0.13741.15340.24781.62810.0446-0.1704-0.09670.1947-0.0426-0.10540.13250.0099-0.002-0.1272-0.03-0.0287-0.14070.0106-0.12570.53823.704-1.169
21.1822-0.3139-0.26291.55710.68351.9551-0.0844-0.18460.04630.1180.03340.01050.02960.00640.051-0.13560.04240.0057-0.12780.0005-0.133221.17945.17622.71
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 300
2X-RAY DIFFRACTION2B1 - 300

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