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- PDB-4p66: Electrostatics of Active Site Microenvironments of E. coli DHFR -

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Basic information

Entry
Database: PDB / ID: 4p66
TitleElectrostatics of Active Site Microenvironments of E. coli DHFR
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / catalysis / electrostatics / catalytic cycle
Function / homology
Function and homology information


glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / metal ion binding
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
METHOTREXATE / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.84 Å
AuthorsLiu, C.T. / Layfield, J.P. / Stewart III, R.J. / French, J.B. / Hanoian, P. / Asbury, J.B. / Hammes-Schiffer, S. / Benkovic, S.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM092946 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM056207 United States
CitationJournal: J.Am.Chem.Soc. / Year: 2014
Title: Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.
Authors: Liu, C.T. / Layfield, J.P. / Stewart, R.J. / French, J.B. / Hanoian, P. / Asbury, J.B. / Hammes-Schiffer, S. / Benkovic, S.J.
History
DepositionMar 21, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 16, 2014Provider: repository / Type: Initial release
Revision 1.1Aug 6, 2014Group: Structure summary
Revision 1.2Oct 1, 2014Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Derived calculations ...Author supporting evidence / Derived calculations / Other / Source and taxonomy
Category: entity_src_gen / pdbx_audit_support ...entity_src_gen / pdbx_audit_support / pdbx_database_status / pdbx_struct_oper_list
Item: _entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization ..._entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization / _pdbx_database_status.pdb_format_compatible / _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / refine_hist / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _refine_hist.pdbx_number_atoms_nucleic_acid / _refine_hist.pdbx_number_atoms_protein / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry
Revision 1.6Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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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)19,2765
Polymers17,9981
Non-polymers1,2784
Water1,58588
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)34.411, 44.861, 98.428
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Dihydrofolate reductase /


Mass: 17998.260 Da / Num. of mol.: 1 / Mutation: T46(XCN), C85A, C152S
Source method: isolated from a genetically manipulated source
Details: Contains a nitrile probe (XCN) attached at position 46
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: folA, ECs0051, LF82_0721 / Production host: Escherichia coli (E. coli) / References: UniProt: C3TR70, dihydrofolate reductase
#2: Chemical ChemComp-MTX / METHOTREXATE / Methotrexate


Mass: 454.439 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H22N8O5 / Comment: chemotherapy*YM
#3: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate


Mass: 743.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H28N7O17P3
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 88 / Source method: isolated from a natural source / Formula: H2O

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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: 2.11 Å3/Da / Density % sol: 41.72 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5 / Details: 100 mM Calcium Acetate 34% PEG 400 100 mM HEPES

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: A1 / Wavelength: 0.978 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jul 12, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978 Å / Relative weight: 1
ReflectionResolution: 1.83→50 Å / Num. obs: 13385 / % possible obs: 97.3 % / Redundancy: 3.9 % / Rmerge(I) obs: 0.107 / Χ2: 1.9 / Net I/av σ(I): 13.971 / Net I/σ(I): 16.7 / Num. measured all: 52399
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2% possible all
1.83-1.863.50.3116401.22993.8
1.86-1.93.70.2666501.37796.4
1.9-1.933.80.2386611.41398.5
1.93-1.9740.2096451.56499.8
1.97-2.0140.1916891.66998
2.01-2.063.90.1756571.89799.8
2.06-2.1140.1586671.90497.9
2.11-2.1740.156641.88999.7
2.17-2.2340.146701.90298
2.23-2.3140.1326652.0299.3
2.31-2.3940.1276632.16597.6
2.39-2.4840.1226722.18797.5
2.48-2.640.1126692.20897.7
2.6-2.7340.1076592.08898.4
2.73-2.940.1026862.36297.7
2.9-3.133.90.0966752.15796.8
3.13-3.4440.0876651.93396
3.44-3.943.90.0836731.83696.4
3.94-4.973.90.0856921.82894.9
4.97-503.60.1187232.13892.3

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Processing

Software
NameVersionClassification
HKL-2000data reduction
REFMAC5.5.0110refinement
PDB_EXTRACT3.14data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GH8

4gh8


Resolution: 1.84→49.21 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.921 / SU B: 2.874 / SU ML: 0.09 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.159 / ESU R Free: 0.145 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2293 668 5 %RANDOM
Rwork0.1881 12684 --
obs0.1901 13352 96.72 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 56.55 Å2 / Biso mean: 17.67 Å2 / Biso min: 10.51 Å2
Baniso -1Baniso -2Baniso -3
1--0.41 Å20 Å20 Å2
2--1.02 Å20 Å2
3----0.61 Å2
Refinement stepCycle: final / Resolution: 1.84→49.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1239 0 83 88 1410
Biso mean--25.4 32.54 -
Num. residues----159
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0221360
X-RAY DIFFRACTIONr_bond_other_d0.0060.02879
X-RAY DIFFRACTIONr_angle_refined_deg1.5312.021858
X-RAY DIFFRACTIONr_angle_other_deg1.0843.0192139
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.6175156
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.70524.03557
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.47915196
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.051157
X-RAY DIFFRACTIONr_chiral_restr0.0930.2200
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0211477
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02269
X-RAY DIFFRACTIONr_mcbond_it0.8841.5801
X-RAY DIFFRACTIONr_mcbond_other0.2141.5321
X-RAY DIFFRACTIONr_mcangle_it1.6721284
X-RAY DIFFRACTIONr_scbond_it2.3023559
X-RAY DIFFRACTIONr_scangle_it3.6324.5574
LS refinement shellResolution: 1.84→1.89 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.302 40 -
Rwork0.198 828 -
all-868 -
obs--86.2 %

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