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- PDB-8ta0: Crystal Structure of Dihydrofolate reductase (DHFR) from Mycobact... -

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Basic information

Entry
Database: PDB / ID: 8ta0
TitleCrystal Structure of Dihydrofolate reductase (DHFR) from Mycobacterium ulcerans Agy99 in complex with NADP and inhibitor MAM881
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 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 / Dihydrofolate reductase
Similarity search - Component
Biological speciesMycobacterium ulcerans (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.95 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
DepositionJun 26, 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

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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)20,2183
Polymers19,0361
Non-polymers1,1822
Water2,414134
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)28.631, 66.290, 44.030
Angle α, β, γ (deg.)90.00, 91.29, 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-DQI / 3-[2-(3-{[(6M)-2,4-diamino-6-(3-methoxyphenyl)pyrimidin-5-yl]oxy}propoxy)phenyl]propanoic acid


Mass: 438.476 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H26N4O5 / Feature type: SUBJECT OF INVESTIGATION
#3: 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
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 134 / 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: 43.95 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Morpheus H4: 12.5%(v/v) MPD, 12.5%(v/v) PEG 1000, 12.5%(w/v) PEG 3350, 100 mM Imidazole/MES, pH 6.5, 20 mM DL-Glutamic acid, 20 mM DL-Alanine; 20 mM Glycine, 20 mM DL-Lysine ...Details: Morpheus H4: 12.5%(v/v) MPD, 12.5%(v/v) PEG 1000, 12.5%(w/v) PEG 3350, 100 mM Imidazole/MES, pH 6.5, 20 mM DL-Glutamic acid, 20 mM DL-Alanine; 20 mM Glycine, 20 mM DL-Lysine monohydrochloride and 20 mM DL-Serine. MyulA.01062.a.B13.PS38720 at 8.9 mg/mL. 2mM MAM881 and 2mM NADP added to the protein prior to crystallization. Plate 13387 well H4 drop 3, 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 20, 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: 1.95→44.02 Å / Num. obs: 12079 / % possible obs: 100 % / Redundancy: 6.8 % / CC1/2: 0.995 / Rmerge(I) obs: 0.122 / Rpim(I) all: 0.05 / Rrim(I) all: 0.132 / Χ2: 1 / Net I/σ(I): 12.4 / Num. measured all: 82492
Reflection shellResolution: 1.95→2 Å / % possible obs: 100 % / Redundancy: 3.8 % / Rmerge(I) obs: 0.71 / Num. measured all: 3300 / Num. unique obs: 858 / CC1/2: 0.649 / Rpim(I) all: 0.418 / Rrim(I) all: 0.828 / Χ2: 0.88 / Net I/σ(I) obs: 2.1

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Processing

Software
NameVersionClassification
PHENIX1.21rc1_4933refinement
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.95→28.62 Å / SU ML: 0.17 / Cross valid method: FREE R-VALUE / σ(F): 1.1 / Phase error: 20.42 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2092 620 5.15 %
Rwork0.1519 --
obs0.1549 12043 99.79 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.95→28.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1252 0 80 134 1466
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0091382
X-RAY DIFFRACTIONf_angle_d1.1251902
X-RAY DIFFRACTIONf_dihedral_angle_d13.166505
X-RAY DIFFRACTIONf_chiral_restr0.056208
X-RAY DIFFRACTIONf_plane_restr0.012239
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.95-2.150.23811460.18192838X-RAY DIFFRACTION100
2.15-2.460.20331500.15552851X-RAY DIFFRACTION100
2.46-3.090.23811750.15662839X-RAY DIFFRACTION100
3.09-28.620.18261490.13732895X-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)
12.1488-0.0752-0.40862.79950.10822.76840.005-0.22140.15010.12970.0072-0.023-0.1937-0.0074-0.02460.04010.00730.00870.0513-0.01410.08364.7408-6.446516.2365
21.83160.61440.21641.24652.20094.3805-0.0588-0.30020.3564-0.0484-0.32960.3943-0.4801-0.51740.0190.11810.0295-0.01010.1457-0.05010.1344-7.6374-6.058713.7813
32.6026-0.6858-0.87762.8068-0.0532.31310.0192-0.15840.0797-0.0286-0.0585-0.10510.0463-0.02650.04830.0328-0.00520.01060.0374-0.00140.061-0.5673-18.425710.8657
42.327-0.0188-0.29721.5497-0.1441.9446-0.1035-0.0911-0.2850.0564-0.0278-0.00320.2069-0.02380.11450.1037-0.01060.01560.08130.00840.1333-2.0198-23.431111.5703
52.1133-1.32510.83323.6248-0.64285.26510.03210.12150.178-0.5312-0.1639-0.11770.03090.44130.06350.11870.00950.02080.0972-0.02840.10473.7938-23.39521.2924
61.58710.2916-1.04113.38911.70171.7578-0.11320.2485-0.14-0.497-0.09680.1073-0.0493-0.31550.13130.1874-0-0.0180.11260.00250.1074-2.8046-16.27870.1135
71.7202-1.0578-0.36071.8257-0.00151.2613-0.00340.12070.05610.0259-0.1625-0.0692-0.18330.04070.05880.0639-0.0236-0.01330.07080.01560.11555.3932-6.78776.3367
83.6043-1.4884-0.1191.8226-0.1750.117-0.2784-0.81040.49870.48350.3896-0.4063-0.08890.3964-0.02480.13570.0083-0.03560.2734-0.05670.180713.621-6.17222.6236
90.0922-0.20810.52150.5307-1.02173.30780.1835-0.02020.21180.0702-0.14750.0416-0.32890.39840.02320.1231-0.02910.02470.11990.01820.1218.9601-0.14483.4988
101.9954-0.1782-2.65651.02140.84316.28130.16170.02690.26640.27220.0479-0.1212-0.2297-0.30190.03240.22780.0224-0.00980.0859-0.01260.1473-0.78513.780112.7604
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 51 )
4X-RAY DIFFRACTION4chain 'A' and (resid 52 through 78 )
5X-RAY DIFFRACTION5chain 'A' and (resid 79 through 87 )
6X-RAY DIFFRACTION6chain 'A' and (resid 88 through 102 )
7X-RAY DIFFRACTION7chain 'A' and (resid 103 through 122 )
8X-RAY DIFFRACTION8chain 'A' and (resid 123 through 132 )
9X-RAY DIFFRACTION9chain 'A' and (resid 133 through 145 )
10X-RAY DIFFRACTION10chain 'A' and (resid 146 through 165 )

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