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- PDB-2w3w: MYCOBACTERIUM AVIUM DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH ... -

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Basic information

Entry
Database: PDB / ID: 2w3w
TitleMYCOBACTERIUM AVIUM DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND A LIPOPHILIC ANTIFOLATE SELECTIVE FOR M. AVIUM DHFR, 6-((2,5- DIETHOXYPHENYL)AMINOMETHYL)-2,4-DIAMINO-5-METHYLPYRIDO(2,3-D) PYRIMIDINE (SRI-8686)
ComponentsDIHYDROFOLATE REDUCTASE
KeywordsOXIDOREDUCTASE / NONCLASSICAL ANTIFOLATES / ONE-CARBON METABOLISM / LIPOPHILIC ANTIFOLATES / NADP / REDUCTASE
Function / homology
Function and homology information


dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / 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-NDP / Chem-VG9 / Dihydrofolate reductase
Similarity search - Component
Biological speciesMYCOBACTERIUM AVIUM (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsLeung, A.K.W. / Reynolds, R.C. / Borhani, D.W.
CitationJournal: To be Published
Title: Structural Basis for Selective Inhibition of Mycobacterium Avium Dihydrofolate Reductase by a Lipophilic Antifolate
Authors: Leung, A.K.W. / Ross, L.J. / Zywno-Van Ginkel, S. / Reynolds, R.C. / Seitz, L.E. / Pathak, V. / Barrow, W.W. / White, E.L. / Suling, W.J. / Piper, J.R. / Borhani, D.W.
History
DepositionNov 16, 2008Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Version format compliance
Revision 1.2Mar 6, 2019Group: Advisory / Data collection ...Advisory / Data collection / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc ...exptl_crystal_grow / pdbx_database_proc / pdbx_database_status / pdbx_unobs_or_zero_occ_atoms
Item: _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Revision 1.3May 8, 2019Group: Data collection / Experimental preparation
Category: database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow
Item: _exptl_crystal_grow.method
Revision 1.4Dec 13, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / 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 / pdbx_unobs_or_zero_occ_atoms / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
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

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Assembly

Deposited unit
A: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,5663
Polymers18,4521
Non-polymers1,1142
Water2,270126
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)36.729, 53.965, 81.191
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 DIHYDROFOLATE REDUCTASE


Mass: 18451.822 Da / Num. of mol.: 1 / Fragment: RESIDUES, 1-167
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) MYCOBACTERIUM AVIUM (bacteria)
Description: STRAIN OBTAINED FROM THE AIDS RESEARCH AND REFERENCE REAGENT PROGRAM, DIVISION OF AIDS, NIAID, NIH, CATALOG NUMBER 1786.
Plasmid: PET11A-MACDHFR-DEL / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: O30463, dihydrofolate reductase
#2: Chemical ChemComp-VG9 / 6-{[(2,5-DIETHOXYPHENYL)AMINO]METHYL}-5-METHYLPYRIDO[2,3-D]PYRIMIDINE-2,4-DIAMINE


Mass: 368.433 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H24N6O2
#3: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 126 / Source method: isolated from a natural source / Formula: H2O
Nonpolymer details6-((2,5-DIETHOXYPHENYL)AMINOMETHYL)-2,4-DIAMINO-5- 6-((2,5-DIETHOXYPHENYL)AMINOMETHYL)-2, 4-DIAMINO- ...6-((2,5-DIETHOXYPHENYL)AMINOMETHYL)-2,4-DIAMINO-5- 6-((2,5-DIETHOXYPHENYL)AMINOMETHYL)-2, 4-DIAMINO-5-METHYLPYRIDO(2,3-D)PYRIMIDINE (VG9): LIGAND IS ALSO KNOWN AS SRI-8686. NOTE THAT THE 2' ETHYL GROUP OF SRI-8686 WAS NOT LOCATED IN ELECTRON DENSITY MAPS. APPROXIMATE COORDINATES FOR THESE TWO ATOMS ARE INCLUDED, BUT AT ZERO OCCUPANCY.
Sequence detailsRESIDUES 168-181 OF WILDTYPE M. AVIUM DHFR WERE DELETED IN THE EXPRESSION CONSTRUCT. THE FIRST ...RESIDUES 168-181 OF WILDTYPE M. AVIUM DHFR WERE DELETED IN THE EXPRESSION CONSTRUCT. THE FIRST ELEVEN CODONS OF THE M. AVIUM DHFR READING FRAME WERE MUTATED TO INCREASE THE A AND T CONTENT. THE CODONS USED WERE 5'-ATG-ACT-CGT-GCT-GAA- GTA-GGT-CTG-GTA-TGG-GCT. PCR INTRODUCED AN UNINTENDED SILENT MUTATION AT VAL120 (GTC MUTATED TO GTT).

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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: 2.18 Å3/Da / Density % sol: 43.7 % / Description: NONE
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: MAVDHFR (15 MG/ML) IN 20 MM HEPES (PH 7.0), 1 MM DTT, 0.1 MM EDTA, 1.5 MM NAN3 WAS MIXED WITH NADPH (3 MM, 15 MIN AT ROOM TEMPERATURE) AND THEN SRI-8686 (2 MM, 15 MIN ON ICE). ...Details: MAVDHFR (15 MG/ML) IN 20 MM HEPES (PH 7.0), 1 MM DTT, 0.1 MM EDTA, 1.5 MM NAN3 WAS MIXED WITH NADPH (3 MM, 15 MIN AT ROOM TEMPERATURE) AND THEN SRI-8686 (2 MM, 15 MIN ON ICE). CRYSTALLIZATION (HANGING DROP VAPOR PHASE EQUILIBRATION) WAS ACHIEVED BY MIXING WITH AN EQUAL VOLUME OF THE PROTEIN COMPLEX WITH A RESERVOIR SOLUTION CONSISTING OF 55-70% 2-METHYL-2, 4-PENTANEDIOL (MPD) AND 100 MM HEPES (PH 6.5-7.5), AND SUSPENDING THE MIXTURE OVER THE RESERVOIR AT 277 K. SMALL ROD-LIKE CRYSTALS (0.01 X 0.01 X 0.05 MM) GREW WITHIN 2 DAYS. CRYSTALS WERE FLASH-COOLED DIRECTLY FROM THE DROP IN LIQUID N2.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 1.1
DetectorType: BRANDEIS UNIVERSITY BRANDEIS B4 / Detector: CCD / Date: Apr 13, 2000 / Details: MIRRORS
RadiationMonochromator: SI / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 1.6→15 Å / Num. obs: 18299 / % possible obs: 83.5 % / Observed criterion σ(I): -10 / Redundancy: 4.5 % / Biso Wilson estimate: 18.7 Å2 / Rmerge(I) obs: 0.08 / Net I/σ(I): 10.8
Reflection shellResolution: 1.6→1.64 Å / Redundancy: 1.2 % / Rmerge(I) obs: 0.46 / Mean I/σ(I) obs: 1.7 / % possible all: 37.9

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Processing

Software
NameVersionClassification
REFMAC5.2.0019refinement
MOSFLMdata reduction
SCALAdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: UNPUBLISHED M. AVIUM DHFR-NADPH- TRIMETHOPRIM COMPLEX, SINCE DEPOSITED AS PDB ENTRY 2W3V.

2w3v


Resolution: 1.6→14.98 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.946 / SU B: 3.279 / SU ML: 0.066 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.11 / ESU R Free: 0.106 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. NOTE THAT THE 2' ETHYL GROUP OF SRI-8686 (VG9) WAS NOT LOCATED IN ELECTRON DENSITY MAPS. APPROXIMATE COORDINATES FOR THESE TWO ATOMS ARE ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. NOTE THAT THE 2' ETHYL GROUP OF SRI-8686 (VG9) WAS NOT LOCATED IN ELECTRON DENSITY MAPS. APPROXIMATE COORDINATES FOR THESE TWO ATOMS ARE INCLUDED, BUT AT ZERO OCCUPANCY.
RfactorNum. reflection% reflectionSelection details
Rfree0.207 920 5 %RANDOM
Rwork0.173 ---
obs0.175 17339 83.3 %-
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: 16.96 Å2
Baniso -1Baniso -2Baniso -3
1-0.89 Å20 Å20 Å2
2---0.2 Å20 Å2
3----0.7 Å2
Refinement stepCycle: LAST / Resolution: 1.6→14.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1247 0 75 126 1448
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0211430
X-RAY DIFFRACTIONr_bond_other_d0.0020.02975
X-RAY DIFFRACTIONr_angle_refined_deg1.6642.0171971
X-RAY DIFFRACTIONr_angle_other_deg1.3132335
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9155179
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.68520.74154
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.81415208
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.9911517
X-RAY DIFFRACTIONr_chiral_restr0.0910.2211
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.021613
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02310
X-RAY DIFFRACTIONr_nbd_refined0.2150.2231
X-RAY DIFFRACTIONr_nbd_other0.2150.21065
X-RAY DIFFRACTIONr_nbtor_refined0.1870.2677
X-RAY DIFFRACTIONr_nbtor_other0.0850.2789
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1370.297
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1750.211
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2770.230
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.2240.218
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.2651.51098
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.43821390
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it2.4083708
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it3.3774.5581
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: 1.6→1.64 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.331 30
Rwork0.228 545
Refinement TLS params.Method: refined / Origin x: 13.892 Å / Origin y: 2.395 Å / Origin z: 46.63 Å
111213212223313233
T-0.0861 Å2-0.0208 Å2-0.0056 Å2--0.0501 Å20.0158 Å2---0.0225 Å2
L1.1401 °2-0.1259 °20.2162 °2-1.5339 °21.0122 °2--3.8496 °2
S0.0235 Å °-0.0003 Å °0.0006 Å °0.0573 Å °0.0297 Å °-0.0504 Å °0.121 Å °-0.1365 Å °-0.0532 Å °

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