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- PDB-2inq: Neutron Crystal Structure of Escherichia coli Dihydrofolate Reduc... -

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Basic information

Entry
Database: PDB / ID: 2inq
TitleNeutron Crystal Structure of Escherichia coli Dihydrofolate Reductase Bound to the Anti-cancer drug, Methotrexate
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / Neutron Structure / Deuterium Exchange / Pseudo-Rossman Fold / Nucleotide Binding Domain / Chemotherapy
Function / homology
Function and homology information


methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process ...methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / NADPH binding / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / response to xenobiotic stimulus / 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-MT1 / Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli K12 (bacteria)
MethodNEUTRON DIFFRACTION / FOURIER SYNTHESIS / Resolution: 2.2 Å
AuthorsBennett, B.C. / Langan, P.A. / Coates, L. / Schoenborn, B. / Dealwis, C.G.
CitationJournal: Proc.Natl.Acad.Sci.Usa / Year: 2006
Title: Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate.
Authors: Bennett, B.C. / Langan, P.A. / Coates, L. / Mustyakimov, M. / Schoenborn, B. / Howell, E.E. / Dealwis, C.G.
History
DepositionOct 8, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 6, 2007Provider: repository / Type: Initial release
Revision 1.1Apr 2, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.3Oct 20, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.4Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dihydrofolate reductase
B: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,9524
Polymers36,0412
Non-polymers9112
Water2,738152
1
A: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,4762
Polymers18,0201
Non-polymers4551
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,4762
Polymers18,0201
Non-polymers4551
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)93.120, 93.120, 73.870
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number169
Space group name H-MP61
DetailsThe asymmetric unit is a crystallographic dimer, containing 2 distinct DHFR monomers. However, E. coli DHFR functions as a monomeric unit.

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Components

#1: Protein Dihydrofolate reductase


Mass: 18020.326 Da / Num. of mol.: 2 / Mutation: N37D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Species: Escherichia coli / Strain: K-12 / Gene: folA / Plasmid: pUC8-ecDHFR / Production host: Escherichia coli (E. coli) / Strain (production host): SK383 / References: UniProt: P0ABQ4, dihydrofolate reductase
#2: Chemical ChemComp-MT1 / N-(4-{[(2,4-DIAMINOPTERIDIN-1-IUM-6-YL)METHYL](METHYL)AMINO}BENZOYL)-L-GLUTAMIC ACID / METHOTREXATE PROTONATED AT N1


Mass: 455.447 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H23N8O5 / Comment: chemotherapy*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 152 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: NEUTRON DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.56 Å3/Da / Density % sol: 52.03 %
Crystal growTemperature: 277 K / pH: 7.5
Details: 16% (v/v) Polyethylene Glycol 400, 0.2 M CaCl2, 0.1 M Na-HEPES (pH = 7.5); complex [] = 50 mg/ml, Microbatch under parrafin oil, temperature 277K, pH 7.50

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Data collection

DiffractionMean temperature: 293 K
Diffraction sourceWavelength: 0.6-7.0
DetectorType: TIME-OF-FLIGHT MULTIWIRE HE3 NEUTRON DETECTOR / Detector: AREA DETECTOR / Date: Feb 15, 2005
RadiationMonochromator: CHOPPER / Protocol: LAUE / Monochromatic (M) / Laue (L): L / Scattering type: neutron
Radiation wavelength
IDWavelength (Å)Relative weight
10.61
271
ReflectionResolution: 2.17→38.6 Å / Num. obs: 14213 / % possible obs: 79.7 % / Observed criterion σ(I): 1.5 / Redundancy: 2.9 % / Rmerge(I) obs: 0.136 / Rsym value: 0.07 / Net I/σ(I): 3.7
Reflection shellResolution: 2.17→2.29 Å / Redundancy: 1.7 % / Mean I/σ(I) obs: 1.7 / Rsym value: 0.325 / % possible all: 63.7

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Processing

Software
NameVersionClassification
d*TREKmodified for Time-of-Flight data collectiondata scaling
SHELXL-97model building
SHELXL-97refinement
d*TREKMODIFIED FOR TIME-OF- FLIGHT SPOT INTEGRATIONdata reduction
CCP4(SCALA)data scaling
SHELXL-97phasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 3DRC (293 K E. COLI DHFR/MTX X-RAY STRUCTURE, 1.7A RESOLUTION)

3drc


Resolution: 2.2→6.5 Å / Cross valid method: THROUGHOUT / σ(F): 3 / Stereochemistry target values: ENGH & HUBER
RfactorNum. reflection% reflectionSelection details
Rfree0.233 702 5.5 %RANDOM
all-14213 --
obs--79.7 %-
Refinement stepCycle: LAST / Resolution: 2.2→6.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2516 0 66 152 2734
Refine LS restraints
Refine-IDTypeDev ideal
NEUTRON DIFFRACTIONs_bond_d0.046
NEUTRON DIFFRACTIONs_angle_d0.048
NEUTRON DIFFRACTIONs_similar_dist
NEUTRON DIFFRACTIONs_from_restr_planes0.016
NEUTRON DIFFRACTIONs_zero_chiral_vol0.059
NEUTRON DIFFRACTIONs_non_zero_chiral_vol0.052
NEUTRON DIFFRACTIONs_anti_bump_dis_restr0.057
NEUTRON DIFFRACTIONs_rigid_bond_adp_cmpnt
NEUTRON DIFFRACTIONs_similar_adp_cmpnt
NEUTRON DIFFRACTIONs_approx_iso_adps

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