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- PDB-8f84: Crystal Structure of Dihydrofolate reductase (DHFR) from Mycobact... -

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Basic information

Entry
Database: PDB / ID: 8f84
TitleCrystal Structure of Dihydrofolate reductase (DHFR) from Mycobacterium ulcerans Agy99 in complex with NADP and inhibitor MAM787
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 reductase / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding
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-XK8 / Dihydrofolate reductase
Similarity search - Component
Biological speciesMycobacterium ulcerans (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.1 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,1833
Polymers19,0361
Non-polymers1,1482
Water3,513195
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)28.698, 66.374, 44.146
Angle α, β, γ (deg.)90.00, 91.44, 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 / 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-XK8 / 3-[2-(3-{[2,4-diamino-6-(4-hydroxybutyl)pyrimidin-5-yl]oxy}propoxy)phenyl]propanoic acid


Mass: 404.460 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28N4O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 195 / 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: 2.21 Å3/Da / Density % sol: 44.29 %
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: 1.1→44.13 Å / Num. obs: 65812 / % possible obs: 98.3 % / Redundancy: 6.6 % / CC1/2: 1 / Rmerge(I) obs: 0.024 / Rpim(I) all: 0.01 / Rrim(I) all: 0.026 / Χ2: 0.96 / Net I/σ(I): 30.2 / Num. measured all: 433825
Reflection shellResolution: 1.1→1.12 Å / % possible obs: 76.2 % / Redundancy: 4.3 % / Rmerge(I) obs: 0.236 / Num. measured all: 10791 / Num. unique obs: 2486 / CC1/2: 0.954 / Rpim(I) all: 0.125 / Rrim(I) all: 0.269 / Χ2: 1 / Net I/σ(I) obs: 5.2

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.1→23.78 Å / SU ML: 0.06 / Cross valid method: FREE R-VALUE / σ(F): 1.21 / Phase error: 12.22 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.144 3297 5.01 %
Rwork0.1254 --
obs0.1263 65775 98.25 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.1→23.78 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1246 0 77 195 1518
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0071458
X-RAY DIFFRACTIONf_angle_d1.0532017
X-RAY DIFFRACTIONf_dihedral_angle_d12.311598
X-RAY DIFFRACTIONf_chiral_restr0.069222
X-RAY DIFFRACTIONf_plane_restr0.013253
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.1-1.120.16181090.13871954X-RAY DIFFRACTION75
1.12-1.130.13851230.12782293X-RAY DIFFRACTION86
1.13-1.150.11471170.1112602X-RAY DIFFRACTION98
1.15-1.170.1381600.10922592X-RAY DIFFRACTION100
1.17-1.190.1421310.10432670X-RAY DIFFRACTION100
1.19-1.210.11841470.11112614X-RAY DIFFRACTION100
1.21-1.230.13831610.1182625X-RAY DIFFRACTION100
1.23-1.260.13071350.11582641X-RAY DIFFRACTION100
1.26-1.290.1241310.10762646X-RAY DIFFRACTION100
1.29-1.320.12361300.10752651X-RAY DIFFRACTION100
1.32-1.350.11461320.10672663X-RAY DIFFRACTION100
1.35-1.390.14431110.10862674X-RAY DIFFRACTION100
1.39-1.430.12711150.10822660X-RAY DIFFRACTION100
1.43-1.470.14371270.11242677X-RAY DIFFRACTION100
1.47-1.530.12391320.11272658X-RAY DIFFRACTION100
1.53-1.590.13381480.11182625X-RAY DIFFRACTION100
1.59-1.660.12191440.11572630X-RAY DIFFRACTION100
1.66-1.750.15551450.12212652X-RAY DIFFRACTION100
1.75-1.860.13711490.12942646X-RAY DIFFRACTION100
1.86-20.13231440.12562652X-RAY DIFFRACTION100
2-2.20.14471550.12712657X-RAY DIFFRACTION100
2.2-2.520.15121300.13142637X-RAY DIFFRACTION100
2.52-3.170.13441600.13792677X-RAY DIFFRACTION100
3.17-23.780.16991610.13552682X-RAY DIFFRACTION100

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