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- PDB-7d6g: Neutron crystal Structure of E.coli Dihydrofolate Reductase compl... -

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Basic information

Entry
Database: PDB / ID: 7d6g
TitleNeutron crystal Structure of E.coli Dihydrofolate Reductase complexed with folate and NADP+ at pH4.5
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / alpha beta alpha sandwich
Function / homology
Function and homology information


methotrexate binding / dihydrofolic acid binding / 10-formyltetrahydrofolate biosynthetic process / response to methotrexate / NADP+ binding / folic acid biosynthetic process / folic acid binding / dihydrofolate metabolic process / folic acid metabolic process / dihydrofolate reductase ...methotrexate binding / dihydrofolic acid binding / 10-formyltetrahydrofolate biosynthetic process / response to methotrexate / NADP+ binding / folic acid biosynthetic process / folic acid binding / dihydrofolate metabolic process / folic acid metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / NADPH binding / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / response to xenobiotic stimulus / response to antibiotic / 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
DEUTERATED WATER / FOLIC ACID / : / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / NEUTRON DIFFRACTION / NUCLEAR REACTOR / MOLECULAR REPLACEMENT / Resolution: 1.65 Å
Model detailsneutron diffraction
AuthorsWan, Q. / Dealwis, C.
Funding support China, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32071264 China
National Science Foundation (NSF, China)31670790 China
CitationJournal: Acs Catalysis / Year: 2021
Title: Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid-Base Catalysis
Authors: Wan, Q. / Bennett, B.C. / Wymore, T. / Li, Z. / Wilson, M.A. / Brooks III, C.L. / Langan, P. / Kovalevsky, A. / Dealwis, C.G.
History
DepositionSep 30, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 9, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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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)19,2594
Polymers18,0191
Non-polymers1,2403
Water2,072115
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, monodisperse
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14560 Å2
ΔGint12 kcal/mol
Surface area8640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)34.102, 45.595, 99.113
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: 18019.340 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: folA, tmrA, b0048, JW0047 / Plasmid: pET-sumo / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABQ4, dihydrofolate reductase
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-FOL / FOLIC ACID


Mass: 441.397 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H19N7O6 / Feature type: SUBJECT OF INVESTIGATION
#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-DOD / water


Mass: 18.015 Da / Num. of mol.: 115 / Source method: isolated from a natural source / Formula: D2O
Has ligand of interestY
Nonpolymer detailsFOL in this structure is the tautomer with double bond between N1-C2 atoms instead of N3-C2 atoms. ...FOL in this structure is the tautomer with double bond between N1-C2 atoms instead of N3-C2 atoms. Thus H atom (H31) was observed on N3 atom.

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Experimental details

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Experiment

Experiment
MethodNumber of used crystals
X-RAY DIFFRACTION1
NEUTRON DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.14 Å3/Da / Density % sol: 42.47 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.5
Details: 40mg/mL DHFR-folate-NADP+ complex, 12% (v/v) PEG 400, 100mM MnCl2, 100mM NaAc, pH4.5

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Data collection

Diffraction
IDMean temperature (K)Crystal-IDSerial crystal experiment
12911N
22911N
Diffraction source
SourceTypeIDWavelength (Å)
ROTATING ANODERIGAKU MICROMAX-00711.54
NUCLEAR REACTOROTHER23.3
Detector
TypeIDDetectorDate
RIGAKU RAXIS IV++1IMAGE PLATEAug 10, 2014
CUSTOM-MADE2IMAGE PLATEMay 15, 2015
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-IDMonochromator
1SINGLE WAVELENGTHMx-ray1
2SINGLE WAVELENGTHMneutron3CHOPPER
Radiation wavelength
IDWavelength (Å)Relative weight
11.541
23.31
31
Reflection

Entry-ID: 7D6G / Rmerge(I) obs: 0.041 / Diffraction-ID: 1

Resolution (Å)Num. obs% possible obs (%)Redundancy (%)Χ2Net I/σ(I)
1.65-401816393.93.90.9624.94
2.1-27.311768191.762.325.3
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsΧ2Diffraction-ID% possible all
1.65-1.753.30.4262.5222300.88177.8
2.1-2.180.3352.35890.9592

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
PHENIX1.17.1_3660refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
Refinement

SU ML: 0.11 / Cross valid method: THROUGHOUT / Method to determine structure: MOLECULAR REPLACEMENT / Phase error: 17.25 / 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-IDBiso max (Å2)Biso mean (Å2)Biso min (Å2)Rfactor RfreeRfactor RworkRfactor obsNum. reflection RfreeNum. reflection RworkNum. reflection obs% reflection Rfree (%)% reflection obs (%)Diffraction-IDσ(F)
1.65-27.31X-RAY DIFFRACTION91.126.36688.650.18620.15710.158590416777176815.1191.7611.38
2.1-26.33NEUTRON DIFFRACTION0.21870.17660.178631670184.572.520
Refinement stepCycle: final / Resolution: 1.65→27.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1268 0 129 346 1743
Biso mean--21.63 45.72 -
Num. residues----159
LS refinement shell

Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-IDNum. reflection allTotal num. of bins used% reflection obs (%)
2.09-2.630.26471310.20932927NEUTRON DIFFRACTION3058264
2.63-26.330.1951850.15943775NEUTRON DIFFRACTION3960280
1.65-1.750.26781160.21842114X-RAY DIFFRACTION2230671
1.75-1.890.18931580.17322809X-RAY DIFFRACTION2967694
1.89-2.080.21421830.16442877X-RAY DIFFRACTION3060697
2.08-2.380.20241410.15542954X-RAY DIFFRACTION3095697
2.38-30.19721650.16232961X-RAY DIFFRACTION3126697
3-27.310.14421410.13983062X-RAY DIFFRACTION3203695

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