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

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Basic information

Entry
Database: PDB / ID: 8tbr
TitleCrystal Structure of Dihydrofolate reductase (DHFR) from Mycobacterium ulcerans Agy99 in complex with NADP and inhibitor MAM758
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE/INHIBITOR / SSGCID / STRUCTURAL GENOMICS / SEATTLE STRUCTURAL GENOMICS CENTER FOR INFECTIOUS DISEASE / Dihydrofolate reductase / 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 / Dihydrofolate reductase
Similarity search - Component
Biological speciesMycobacterium ulcerans (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.75 Å
AuthorsSeattle Structural Genomics Center for Infectious Disease / Seattle Structural Genomics Center for Infectious Disease (SSGCID)
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N93022C00036 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
DepositionJun 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 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 1.2Nov 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

Downloads & links

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Assembly

Deposited unit
A: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,1873
Polymers19,0361
Non-polymers1,1522
Water2,774154
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)28.606, 66.332, 43.973
Angle α, β, γ (deg.)90.00, 91.16, 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) / Strain (production host): BL21(DE3) / 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-HIJ / 3-(2-{3-[(2,4-diamino-6-phenylpyrimidin-5-yl)oxy]propoxy}phenyl)propanoic acid


Mass: 408.450 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H24N4O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 154 / 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: 2.19 Å3/Da / Density % sol: 43.87 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Morpheus E4: 12.5%(v/v) MPD, 12.5%(v/v) PEG 1000, 12.5%(w/v) PEG 3350, 100 mM Imidazole/MES, pH 6.5, 30 mM Diethylene glycol, 30 mM Triethyleneglycol, 30 mM Tetraethylene glycol and 30 mM ...Details: Morpheus E4: 12.5%(v/v) MPD, 12.5%(v/v) PEG 1000, 12.5%(w/v) PEG 3350, 100 mM Imidazole/MES, pH 6.5, 30 mM Diethylene glycol, 30 mM Triethyleneglycol, 30 mM Tetraethylene glycol and 30 mM Pentaethylene glycol. MyulA.01062.a.B13.PS38720 at 8.9 mg/mL. 2mM MAM758 and 2mM NADP added to the protein prior to crystallization. Plate 13387 well E4 drop 2, Cryo: direct

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SEALED TUBE / Type: BRUKER D8 QUEST / Wavelength: 1.5418 Å
DetectorType: Bruker PHOTON III / Detector: PIXEL / Date: Jun 26, 2023
RadiationMonochromator: HELIOS MULTILAYER / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.75→43.95 Å / Num. obs: 16609 / % possible obs: 99.9 % / Redundancy: 12.4 % / CC1/2: 0.997 / Rmerge(I) obs: 0.118 / Rpim(I) all: 0.035 / Rrim(I) all: 0.123 / Χ2: 0.92 / Net I/σ(I): 18.1 / Num. measured all: 205983
Reflection shellResolution: 1.75→1.78 Å / % possible obs: 99.6 % / Redundancy: 8.5 % / Rmerge(I) obs: 1.404 / Num. measured all: 7701 / Num. unique obs: 911 / CC1/2: 0.48 / Rpim(I) all: 0.51 / Rrim(I) all: 1.504 / Χ2: 0.77 / Net I/σ(I) obs: 1.5

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Processing

Software
NameVersionClassification
PHENIX1.21rc1_4933refinement
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.75→24.2 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 1.01 / Phase error: 20.67 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2037 788 4.75 %
Rwork0.161 --
obs0.163 16573 99.76 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.75→24.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1252 0 78 154 1484
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011428
X-RAY DIFFRACTIONf_angle_d1.0671972
X-RAY DIFFRACTIONf_dihedral_angle_d13.463537
X-RAY DIFFRACTIONf_chiral_restr0.055214
X-RAY DIFFRACTIONf_plane_restr0.014252
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.75-1.860.29981140.2572605X-RAY DIFFRACTION99
1.86-20.2671510.19782613X-RAY DIFFRACTION100
2-2.20.18621330.16782629X-RAY DIFFRACTION100
2.2-2.520.2151310.15292610X-RAY DIFFRACTION100
2.52-3.180.21041060.14942680X-RAY DIFFRACTION100
3.18-24.20.16691530.13722648X-RAY DIFFRACTION100
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)
12.04970.0155-0.28182.56650.16862.6650.0166-0.27070.19410.16970.0263-0.0671-0.32840.11990.08260.083-0.00530.00180.0899-0.02430.10914.2178-6.595316.5709
21.0384-0.4288-1.08151.99812.17633.3465-0.1192-0.4510.216-0.0872-0.23950.2728-0.5836-0.6452-0.11940.12890.046-0.02060.202-0.07060.1228-8.0038-5.943513.5228
32.2497-0.9491-0.04182.4695-0.30572.7711-0.0834-0.1328-0.10820.17210.04420.16220.0648-0.12910.05550.082-0.00660.02690.0929-0.01350.1037-3.1933-18.681712.925
43.1507-0.5195-0.1272.9418-0.41952.431-0.0879-0.0202-0.112-0.0172-0.04-0.15950.17260.00550.07680.1283-0.00890.03290.0985-0.00540.14171.7595-26.8578.8617
50.7053-0.4527-0.09323.79690.4062.8556-0.00950.103-0.0258-0.53950.03660.08440.0486-0.07250.05780.1253-0.01540.00190.12120.00990.1277-0.2985-19.00970.5601
61.9197-0.78590.03222.1246-0.10071.8595-0.05750.12940.08230.0249-0.126-0.1748-0.35240.1054-0.0310.103-0.0152-0.00920.08740.01590.10555.2956-6.04397.0948
72.4814-0.5936-0.04641.27120.68642.2141-0.1409-0.70380.31380.1830.3556-0.5063-0.08390.3033-0.00880.151-0.0014-0.00670.3005-0.06370.203513.7445-6.271222.7711
80.20620.02060.06291.4876-0.55222.17430.08340.05920.11730.0773-0.0688-0.2141-0.52740.32850.08980.1565-0.02920.0430.15540.03180.12718.53260.15423.4262
91.5303-0.1775-1.30691.66130.56164.26420.070.08380.11250.2432-0.0558-0.1269-0.5746-0.44650.04860.27940.0613-0.0160.115-0.01140.142-0.92023.746112.682
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 26 )
2X-RAY DIFFRACTION2chain 'A' and (resid 27 through 37 )
3X-RAY DIFFRACTION3chain 'A' and (resid 38 through 62 )
4X-RAY DIFFRACTION4chain 'A' and (resid 63 through 78 )
5X-RAY DIFFRACTION5chain 'A' and (resid 79 through 102 )
6X-RAY DIFFRACTION6chain 'A' and (resid 103 through 122 )
7X-RAY DIFFRACTION7chain 'A' and (resid 123 through 132 )
8X-RAY DIFFRACTION8chain 'A' and (resid 133 through 145 )
9X-RAY DIFFRACTION9chain 'A' and (resid 146 through 165 )

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