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- PDB-4pdj: Neutron crystal Structure of E.coli Dihydrofolate Reductase compl... -

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Basic information

Entry
Database: PDB / ID: 4pdj
TitleNeutron crystal Structure of E.coli Dihydrofolate Reductase complexed with folate and NADP+
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / alpha beta alpha sandwich
Function / homology
Function and homology information


methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / NADPH binding ...methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic 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
DIHYDROFOLIC ACID / DEUTERATED WATER / : / Chem-NDP / Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / NEUTRON DIFFRACTION / NUCLEAR REACTOR / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.599 Å
Model detailsneutron diffraction
AuthorsWan, Q. / Kovalevsky, A.Y. / Wilson, M. / Langan, P. / Dealwis, C. / Bennett, B.
Funding support United States, China, 5items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)FWP ERKP752 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071939 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM092999 United States
Yangzhou University2013CXJ083 China
State Education Ministry China
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2014
Title: Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography.
Authors: Wan, Q. / Bennett, B.C. / Wilson, M.A. / Kovalevsky, A. / Langan, P. / Howell, E.E. / Dealwis, C.
History
DepositionApr 18, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 15, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 20, 2016Group: Data collection
Revision 1.2Sep 20, 2017Group: Author supporting evidence / Derived calculations / Source and taxonomy
Category: entity_src_gen / pdbx_audit_support / pdbx_struct_oper_list
Item: _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.3Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 27, 2023Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / 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.ptnr2_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.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_label_alt_id / _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 1.5Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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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)19,3185
Polymers18,0191
Non-polymers1,2994
Water2,144119
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14400 Å2
ΔGint10 kcal/mol
Surface area9220 Å2
MethodPISA
Unit cell
Length a, b, c (Å)34.293, 45.625, 98.974
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Detailsbiological unit is the same as asym.

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Components

#1: Protein Dihydrofolate reductase


Mass: 18019.340 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: folA, tmrA, b0048, JW0047 / Plasmid: pET-sumo / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0ABQ4, dihydrofolate reductase
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-DHF / DIHYDROFOLIC ACID


Mass: 443.413 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H21N7O6
#4: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#5: Chemical ChemComp-DOD / water


Mass: 18.015 Da / Num. of mol.: 119 / Source method: isolated from a natural source / Formula: D2O

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Experimental details

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Experiment

Experiment
MethodNumber of used crystals
X-RAY DIFFRACTION1
NEUTRON DIFFRACTION2

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Sample preparation

CrystalDensity Matthews: 2.15 Å3/Da / Density % sol: 42.75 % / Description: rectangular 3.6 mm3 size crystal
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 40mg/mL DHFR-folate-NADP+ complex, 12% (v/v) PEG400, 100 mM MnCl2, 20 mM imidazole, pH7.0
Temp details: room temperature

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Data collection

Diffraction
IDMean temperature (K)Ambient temp detailsCrystal-ID
1291in capillary1
2291in capillary1
Diffraction source
SourceTypeIDWavelength (Å)Wavelength
ROTATING ANODERIGAKU11.54
NUCLEAR REACTOROTHER23.33.3-4.5
Detector
TypeIDDetectorDateDetails
RIGAKU1IMAGE PLATESep 10, 2013mirrors
CUSTOM-MADE2Aug 10, 2013mirrors
Radiation
IDMonochromatorProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1Multilayer mirrorSINGLE WAVELENGTHMx-ray1
2CHOPPERLAUELneutron2
Radiation wavelength
IDWavelength (Å)Relative weight
11.541
23.31
34.51
Reflection

Entry-ID: 4PDJ

Resolution (Å)Num. obs% possible obs (%)Observed criterion σ(I)Redundancy (%)Rsym valueDiffraction-IDNet I/σ(I)
1.599-302075179.33.27.50.18815.3
1.994-32.48745701.221.2
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsDiffraction-ID% possible all
1.599-1.75.80.313.2161.3
1.994-2.12

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX(phenix.refine: dev_1358)refinement
HKL-3000data processing
Cootmodel building
PDB_EXTRACT3.14data extraction
Cootmodel building
SCALAdata reduction
PHASERphasing
SCALAdata scaling
LAUEGENdata reduction
Refinement

Cross valid method: FREE R-VALUE / Method to determine structure: MOLECULAR REPLACEMENT / Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Starting model: 1RX2

1rx2

/ Stereochemistry target values: ML / Solvent model: FLAT BULK SOLVENT MODEL

Resolution (Å)Refine-IDRfactor RfreeRfactor RworkRfactor obsNum. reflection RfreeNum. reflection obs% reflection Rfree (%)% reflection obs (%)SU MLDiffraction-IDPhase error
1.599-27.413X-RAY DIFFRACTION0.2180.19370.19481068207515.1597.580.18122.64
1.994-32.403NEUTRON DIFFRACTION0.27070.23030.232242787434.8878.420.27225.03
Refinement stepCycle: LAST / Resolution: 1.599→27.413 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1268 0 82 119 1469
Refine LS restraints
Refine-IDTypeDev idealNumber
NEUTRON DIFFRACTIONf_bond_d0.0923166
NEUTRON DIFFRACTIONf_angle_d0.9155398
NEUTRON DIFFRACTIONf_dihedral_angle_d19.963818
NEUTRON DIFFRACTIONf_chiral_restr0.039198
NEUTRON DIFFRACTIONf_plane_restr0.028601
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-IDTotal num. of bins used% reflection obs (%)
1.5991-1.67190.28631420.25332316X-RAY DIFFRACTION894
1.6719-1.760.32171260.24642374X-RAY DIFFRACTION896
1.76-1.87030.25011390.23162392X-RAY DIFFRACTION897
1.8703-2.01470.26541460.21392410X-RAY DIFFRACTION897
2.0147-2.21730.25241350.20922444X-RAY DIFFRACTION898
2.2173-2.5380.23231190.20392508X-RAY DIFFRACTION899
2.538-3.19680.20941390.19922538X-RAY DIFFRACTION8100
3.1968-27.4170.161220.15912701X-RAY DIFFRACTION8100
1.9937-2.28210.33411150.29662226NEUTRON DIFFRACTION364
2.2821-2.8750.28731520.25032700NEUTRON DIFFRACTION378
2.875-32.40740.23271600.19253390NEUTRON DIFFRACTION393

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