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- PDB-2lf1: Solution structure of L. casei dihydrofolate reductase complexed ... -

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Basic information

Entry
Database: PDB / ID: 2lf1
TitleSolution structure of L. casei dihydrofolate reductase complexed with NADPH, 30 structures
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / DHFR / positive cooperativity / protein-ligand interactions
Function / homology
Function and homology information


response to methotrexate / dihydrofolate metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / response to antibiotic / cytosol
Similarity search - Function
Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase-like domain superfamily / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-NDP / Dihydrofolate reductase
Similarity search - Component
Biological speciesLactobacillus casei (bacteria)
MethodSOLUTION NMR / simulated annealing
Model detailsclosest to the average, model 1
AuthorsPolshakov, V. / Feeney, J. / Birdsall, B. / Kovalevskaya, N.
CitationJournal: Biochemistry / Year: 2011
Title: NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, ...Title: NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, conformational changes, and interligand hydrophobic interactions
Authors: Feeney, J. / Birdsall, B. / Kovalevskaya, N.V. / Smurnyy, Y.D. / Navarro Peran, E.M. / Polshakov, V.I.
History
DepositionJun 28, 2011Deposition site: BMRB / Processing site: RCSB
Revision 1.0Dec 21, 2011Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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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)19,0772
Polymers18,3321
Non-polymers7451
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)30 / 50structures with the least restraint violations
RepresentativeModel #1closest to the average

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Components

#1: Protein Dihydrofolate reductase


Mass: 18331.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus casei (bacteria) / Gene: folA, dhfR / Production host: Escherichia coli (E. coli) / Strain (production host): NF1 / References: UniProt: P00381, dihydrofolate reductase
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1132D 1H-15N HSQC
2232D 1H-15N HSQC
1322D 1H-13C HSQC aliphatic
1422D 1H-13C HSQC aromatic
2542D DQF-COSY
1613D CBCA(CO)NH
1713D HN(CA)CB
1813D HNCO
1933D HNHA
11033D HNHB
11123D (H)CCH-TOCSY
11233D 1H-15N NOESY
11323D 1H-13C NOESY
21432D 15N-rejected NOESY

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Sample preparation

Details
Solution-IDContentsSolvent system
12 mM [U-99% 13C; U-99% 15N] DHFR, 2 mM NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, 100 mM potassium chloride, 50 mM potassium phosphate, 95% H2O/5% D2O95% H2O/5% D2O
22 mM [U-99% 13C; U-99% 15N] DHFR, 2 mM NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, 100 mM potassium chloride, 50 mM potassium phosphate, 100% D2O100% D2O
32 mM [U-99% 15N] DHFR, 2 mM NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, 100 mM potassium chloride, 50 mM potassium phosphate, 95% H2O/5% D2O95% H2O/5% D2O
44 mM DHFR, 4 mM NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, 100 mM potassium chloride, 50 mM potassium phosphate, 100% D2O100% D2O
51 mM [U-99% 15N] DHFR, 1 mM NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, 100 mM potassium chloride, 50 mM potassium phosphate, 5 v/v n-octylpenta-ethylene glycol (C8E5), 1.5 v/v n-octanol, 95% H2O/5% D2O95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
2 mMDHFR-1[U-99% 13C; U-99% 15N]1
2 mMNADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE-21
100 mMpotassium chloride-31
50 mMpotassium phosphate-41
2 mMDHFR-5[U-99% 13C; U-99% 15N]2
2 mMNADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE-62
100 mMpotassium chloride-72
50 mMpotassium phosphate-82
2 mMDHFR-9[U-99% 15N]3
2 mMNADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE-103
100 mMpotassium chloride-113
50 mMpotassium phosphate-123
4 mMDHFR-134
4 mMNADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE-144
100 mMpotassium chloride-154
50 mMpotassium phosphate-164
1 mMDHFR-17[U-99% 15N]5
1 mMNADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE-185
100 mMpotassium chloride-195
50 mMpotassium phosphate-205
5 v/vn-octylpenta-ethylene glycol (C8E5)-215
1.5 v/vn-octanol-225
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
1100 6.5 ambient 308 K
2100 6.5 ambient 288 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Varian UnityVarianUNITY5001
Varian INOVAVarianINOVA6002
Varian INOVAVarianINOVA8003
Bruker AvanceBrukerAVANCE6004

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Processing

NMR software
NameDeveloperClassification
VNMRVariancollection
TopSpinBruker Biospincollection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SparkyGoddarddata analysis
AnglesearchPolshakov, Feeneydata analysis
AnglesearchPolshakov, Feeneygeometry optimization
NMRestPolshakov, Feeneydata analysis
Insight IIAccelrys Software Inc.data analysis
CNSBrunger, Adams, Clore, Gros, Nilges and Readstructure solution
ProcheckNMRLaskowski and MacArthurdata analysis
CNSBrunger, Adams, Clore, Gros, Nilges and Readrefinement
RefinementMethod: simulated annealing / Software ordinal: 1
NMR constraintsNOE constraints total: 2071 / NOE intraresidue total count: 802 / NOE long range total count: 416 / NOE medium range total count: 267 / NOE sequential total count: 523 / Hydrogen bond constraints total count: 171
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 50 / Conformers submitted total number: 30 / Maximum lower distance constraint violation: 0 Å / Maximum torsion angle constraint violation: 2 ° / Maximum upper distance constraint violation: 0.1 Å
NMR ensemble rmsDistance rms dev: 0.003 Å / Distance rms dev error: 0.0002 Å

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