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- PDB-8f82: Crystal Structure of Dihydrofolate reductase (DHFR) from Mycobact... -

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Basic information

Entry
Database: PDB / ID: 8f82
TitleCrystal Structure of Dihydrofolate reductase (DHFR) from Mycobacterium ulcerans Agy99 in complex with NADP and inhibitor MAM845
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
BROMIDE ION / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Chem-XJT / Dihydrofolate reductase
Similarity search - Component
Biological speciesMycobacterium ulcerans (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.2 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)20,4545
Polymers19,0361
Non-polymers1,4184
Water3,531196
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)28.666, 66.739, 44.025
Angle α, β, γ (deg.)90.00, 90.48, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 1 molecules A

#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
Details: MAHHHHHHMTSVGLIWAQSTSGVIGRDGGIPWRLPEDLAHFKRLTMGHTVVMGRRTWDSLPAAHRPLPGRRNVVVTRQTG LVAHGAQVVGSLEQALSPAEPDAATWVIGGAQIYALALPLANRCEVTEVDVDLPPEDEDALAPVLDQTWAGTSGEWLVSR SGLRYRMHSYRRL
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

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Non-polymers , 5 types, 200 molecules

#2: Chemical ChemComp-XJT / 3-{2-[3-({(6M)-2,4-diamino-6-[3-(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
#3: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#4: 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
#5: Chemical ChemComp-BR / BROMIDE ION


Mass: 79.904 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Br
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 196 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 44.4 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Morpheus B4: 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 NaF, 30 mM NaBr and 30 mM NaI, MyulA.01062.a.B13.PS38558 at 10 mg/mL. Tray: ...Details: Morpheus B4: 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 NaF, 30 mM NaBr and 30 mM NaI, MyulA.01062.a.B13.PS38558 at 10 mg/mL. Tray: Tray358B4 MAM777, Puck: PUCK HR00407 pin15, Cryo: Direct

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 S 2M / Detector: PIXEL / Date: Sep 22, 2022
RadiationMonochromator: Double Crystal Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.2→44.02 Å / Num. obs: 47045 / % possible obs: 91 % / Redundancy: 4.1 % / CC1/2: 0.999 / Rmerge(I) obs: 0.028 / Rpim(I) all: 0.015 / Rrim(I) all: 0.031 / Χ2: 1.02 / Net I/σ(I): 28.8 / Num. measured all: 193506
Reflection shellResolution: 1.2→1.23 Å / % possible obs: 44.9 % / Redundancy: 2.5 % / Rmerge(I) obs: 0.128 / Num. measured all: 4307 / Num. unique obs: 1711 / CC1/2: 0.971 / Rpim(I) all: 0.096 / Rrim(I) all: 0.161 / Χ2: 0.96 / Net I/σ(I) obs: 7

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.2→44.02 Å / SU ML: 0.09 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 15.66 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1637 2290 4.87 %
Rwork0.1423 --
obs0.1434 47016 90.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.2→44.02 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1296 0 43 196 1535
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0061701
X-RAY DIFFRACTIONf_angle_d0.9622365
X-RAY DIFFRACTIONf_dihedral_angle_d13.117687
X-RAY DIFFRACTIONf_chiral_restr0.076257
X-RAY DIFFRACTIONf_plane_restr0.01305
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.2-1.230.1857910.15681316X-RAY DIFFRACTION43
1.23-1.250.1855740.14271822X-RAY DIFFRACTION59
1.25-1.290.18391430.13812250X-RAY DIFFRACTION75
1.29-1.320.16981280.132717X-RAY DIFFRACTION88
1.32-1.360.16871660.12722886X-RAY DIFFRACTION95
1.36-1.40.15671390.12682983X-RAY DIFFRACTION98
1.4-1.450.15421710.1293051X-RAY DIFFRACTION99
1.45-1.510.1671440.13333102X-RAY DIFFRACTION100
1.51-1.580.1571370.13053061X-RAY DIFFRACTION100
1.58-1.660.16121680.13753038X-RAY DIFFRACTION100
1.66-1.770.15481470.14163085X-RAY DIFFRACTION100
1.77-1.90.17341320.14513107X-RAY DIFFRACTION100
1.91-2.10.17741710.14873047X-RAY DIFFRACTION100
2.1-2.40.16431530.153075X-RAY DIFFRACTION99
2.4-3.020.16371680.15813059X-RAY DIFFRACTION99
3.02-44.020.1551580.13813127X-RAY DIFFRACTION99

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