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- PDB-8f85: Crystal Structure of Dihydrofolate reductase (DHFR) from Mycobact... -

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Basic information

Entry
Database: PDB / ID: 8f85
TitleCrystal Structure of Dihydrofolate reductase (DHFR) from Mycobacterium ulcerans Agy99 in complex with NADP and inhibitor MAM851
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE/INHIBITOR / SSGCID / STRUCTURAL GENOMICS / SEATTLE STRUCTURAL GENOMICS CENTER FOR INFECTIOUS DISEASE / OXIDOREDUCTASE-OXIDOREDUCTASE INHIBITOR complex / OXIDOREDUCTASE / OXIDOREDUCTASE-INHIBITOR complex
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 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
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Chem-XKI / Dihydrofolate reductase
Similarity search - Component
Biological speciesMycobacterium ulcerans (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.15 Å
AuthorsSeattle Structural Genomics Center for Infectious Disease / Seattle Structural Genomics Center for Infectious Disease (SSGCID)
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N93022C00036 United States
National Institutes of Health/Office of the DirectorS10OD030394 United States
CitationJournal: J.Med.Chem. / Year: 2024
Title: Rational Exploration of 2,4-Diaminopyrimidines as DHFR Inhibitors Active against Mycobacterium abscessus and Mycobacterium avium , Two Emerging Human Pathogens.
Authors: Andrade Meirelles, M. / Almeida, V.M. / Sullivan, J.R. / de Toledo, I. / Dos Reis, C.V. / Cunha, M.R. / Zigweid, R. / Shim, A. / Sankaran, B. / Woodward, E.L. / Seibold, S. / Liu, L. / Mian, ...Authors: Andrade Meirelles, M. / Almeida, V.M. / Sullivan, J.R. / de Toledo, I. / Dos Reis, C.V. / Cunha, M.R. / Zigweid, R. / Shim, A. / Sankaran, B. / Woodward, E.L. / Seibold, S. / Liu, L. / Mian, M.R. / Battaile, K.P. / Riley, J. / Duncan, C. / Simeons, F.R.C. / Ferguson, L. / Joji, H. / Read, K.D. / Lovell, S. / Staker, B.L. / Behr, M.A. / Pilli, R.A. / Counago, R.M.
History
DepositionNov 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: diffrn / Item: _diffrn.ambient_temp
Revision 1.2Nov 6, 2024Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _pdbx_entry_details.has_protein_modification
Revision 1.3Nov 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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)20,2553
Polymers19,0361
Non-polymers1,2202
Water2,702150
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)28.751, 66.156, 44.042
Angle α, β, γ (deg.)90.00, 91.73, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Dihydrofolate reductase


Mass: 19035.619 Da / Num. of mol.: 1 / Fragment: MyulA.01062.a.B13 / Mutation: C89S/E96A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium ulcerans (bacteria) / Strain: Agy99 / Gene: dfrA, MUL_2179 / Plasmid: MyulA.01062.a.B13 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0PQG8, dihydrofolate reductase
#2: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H28N7O17P3
#3: Chemical ChemComp-XKI / 3-{2-[3-({(6M)-2,4-diamino-6-[2-(trifluoromethyl)phenyl]pyrimidin-5-yl}oxy)propoxy]phenyl}propanoic acid


Mass: 476.448 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H23F3N4O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 150 / 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: 2.2 Å3/Da / Density % sol: 44.07 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Morpheus F4: 12.5% PDB 1000, 12.5% PEG 3350, 12.5% MPD 0.02M of each monosaccharide (d-glucose, d-mannose, d-galactose, l-fucose, d-xylose, N-acetyld-glucosamine), 0.1M MES/imidazole pH 6.5 ...Details: Morpheus F4: 12.5% PDB 1000, 12.5% PEG 3350, 12.5% MPD 0.02M of each monosaccharide (d-glucose, d-mannose, d-galactose, l-fucose, d-xylose, N-acetyld-glucosamine), 0.1M MES/imidazole pH 6.5 , MyulA.01062.a.B13.PS38558 at 10 mg/mL. Tray: Tray 355D3/ MAM738 +NADP, Puck: CPS5110_06, Cryo: Direct

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 19-ID / Wavelength: 0.97949 Å
DetectorType: DECTRIS EIGER2 XE 9M / Detector: PIXEL / Date: Oct 30, 2022
RadiationMonochromator: Double Crystal Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97949 Å / Relative weight: 1
ReflectionResolution: 1.15→44.02 Å / Num. obs: 54126 / % possible obs: 92.9 % / Redundancy: 4.3 % / CC1/2: 1 / Rmerge(I) obs: 0.022 / Rpim(I) all: 0.012 / Rrim(I) all: 0.025 / Χ2: 0.98 / Net I/σ(I): 27.3 / Num. measured all: 232485
Reflection shellResolution: 1.15→1.17 Å / % possible obs: 59.4 % / Redundancy: 3.5 % / Rmerge(I) obs: 0.205 / Num. measured all: 5966 / Num. unique obs: 1685 / CC1/2: 0.967 / Rpim(I) all: 0.12 / Rrim(I) all: 0.239 / Χ2: 0.99 / Net I/σ(I) obs: 5.7

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.15→23.74 Å / SU ML: 0.08 / Cross valid method: FREE R-VALUE / σ(F): 1.03 / Phase error: 11.51 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1309 2741 5.07 %
Rwork0.1238 --
obs0.1241 54091 92.68 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.15→23.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1328 0 0 150 1478
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0071393
X-RAY DIFFRACTIONf_angle_d1.0511924
X-RAY DIFFRACTIONf_dihedral_angle_d13.28548
X-RAY DIFFRACTIONf_chiral_restr0.072213
X-RAY DIFFRACTIONf_plane_restr0.011242
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.15-1.170.1414910.13051607X-RAY DIFFRACTION59
1.17-1.190.12731020.12081997X-RAY DIFFRACTION72
1.19-1.210.13021330.11992437X-RAY DIFFRACTION89
1.21-1.240.13921480.11412539X-RAY DIFFRACTION92
1.24-1.270.12911430.11052596X-RAY DIFFRACTION94
1.27-1.30.12611450.11092599X-RAY DIFFRACTION94
1.3-1.330.1331440.10932598X-RAY DIFFRACTION94
1.33-1.360.13591290.10592611X-RAY DIFFRACTION94
1.36-1.40.11941470.10132618X-RAY DIFFRACTION96
1.4-1.450.10761310.09922661X-RAY DIFFRACTION95
1.45-1.50.11191330.10242664X-RAY DIFFRACTION96
1.5-1.560.11571260.10242671X-RAY DIFFRACTION96
1.56-1.630.11241600.10832647X-RAY DIFFRACTION96
1.63-1.720.12781410.11032694X-RAY DIFFRACTION97
1.72-1.830.12821410.11812691X-RAY DIFFRACTION97
1.83-1.970.13471560.11142690X-RAY DIFFRACTION98
1.97-2.160.13211560.12272725X-RAY DIFFRACTION98
2.16-2.480.12511470.12862717X-RAY DIFFRACTION98
2.48-3.120.1311370.13692756X-RAY DIFFRACTION98
3.12-23.740.14361310.13732832X-RAY DIFFRACTION99

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