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- PDB-4gh8: Crystal structure of a 'humanized' E. coli dihydrofolate reductase -

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Basic information

Entry
Database: PDB / ID: 4gh8
TitleCrystal structure of a 'humanized' E. coli dihydrofolate reductase
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / tetrahydrofolate / enzyme catalysis / evolution / Dihydrofolate reductase
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 / folic acid metabolic process / dihydrofolate reductase activity ...methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic process / glycine biosynthetic process / dihydrofolate reductase / folic acid metabolic process / 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, 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 / Chem-NDP / Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.85 Å
AuthorsFrench, J.B. / Liu, C.T. / Hanoian, P. / Pringle, T.H. / Hammes-Schiffer, S. / Benkovic, S.J.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2013
Title: Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans.
Authors: Liu, C.T. / Hanoian, P. / French, J.B. / Pringle, T.H. / Hammes-Schiffer, S. / Benkovic, S.J.
History
DepositionAug 7, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2013Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2013Group: Database references
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.3Feb 28, 2024Group: 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 / struct_site
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_asym_id / _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_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _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.ptnr1_auth_asym_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_asym_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_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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)39,2299
Polymers36,7092
Non-polymers2,5207
Water3,045169
1
B: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,5954
Polymers18,3551
Non-polymers1,2403
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
A: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,6355
Polymers18,3551
Non-polymers1,2804
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)52.245, 63.773, 62.439
Angle α, β, γ (deg.)90.00, 106.82, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Dihydrofolate reductase /


Mass: 18354.734 Da / Num. of mol.: 2 / Mutation: N23PP, G51PEKN
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: folA, tmrA, b0048, JW0047 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABQ4, dihydrofolate reductase
#2: Chemical ChemComp-MTX / METHOTREXATE / Methotrexate


Mass: 454.439 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H22N8O5 / Comment: chemotherapy*YM
#3: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate


Mass: 745.421 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 169 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTWO INSERTIONS COMPARING TO UNP P0ABQ4 SEQUENCE, RESIDUE ASN 23 WAS REPLACED BY TWO AMINO ACIDS ...TWO INSERTIONS COMPARING TO UNP P0ABQ4 SEQUENCE, RESIDUE ASN 23 WAS REPLACED BY TWO AMINO ACIDS (PRO PRO) AND RESIDUES GLY 51 WAS REPLACED BY FOUR AMINO ACIDS (PRO GLU LYS ASN).

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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: 2.71 Å3/Da / Density % sol: 54.65 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 200 mM Calcium Acetate 38% Peg 400, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: A1 / Wavelength: 0.987 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jun 24, 2012
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionRedundancy: 2.6 % / Av σ(I) over netI: 12.1 / Number: 83707 / Rmerge(I) obs: 0.078 / Χ2: 1.23 / D res high: 1.85 Å / D res low: 50 Å / Num. obs: 31876 / % possible obs: 95.2
Diffraction reflection shell
Highest resolution (Å)Lowest resolution (Å)% possible obs (%)IDRmerge(I) obsChi squaredRedundancy
5.025096.410.0511.6052.6
3.995.0298.610.0531.532.6
3.483.9998.910.0561.5462.6
3.163.4899.110.0591.4862.6
2.943.1698.910.0691.6382.6
2.762.9498.610.0761.5132.7
2.632.7698.410.0841.4962.6
2.512.6398.510.0941.3042.6
2.412.5198.610.11.2542.7
2.332.4198.410.111.1622.7
2.262.339810.1281.1772.6
2.192.2698.110.1311.132.6
2.142.1997.310.151.0562.6
2.082.1496.510.1621.072.6
2.042.0894.910.1721.0792.6
1.992.0492.810.1810.9282.6
1.951.9989.610.2220.9092.6
1.921.9587.310.250.8362.6
1.881.9284.210.2960.7682.7
1.851.8880.510.3270.742.6
ReflectionResolution: 1.85→50 Å / Num. all: 83707 / Num. obs: 31876 / % possible obs: 95.2 % / Redundancy: 2.6 % / Rmerge(I) obs: 0.078 / Χ2: 1.227 / Net I/σ(I): 8.9
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
1.85-1.882.60.32713500.74180.5
1.88-1.922.70.29614010.768184.2
1.92-1.952.60.2514350.836187.3
1.95-1.992.60.22215150.909189.6
1.99-2.042.60.18115430.928192.8
2.04-2.082.60.17215791.079194.9
2.08-2.142.60.16215841.07196.5
2.14-2.192.60.1516331.056197.3
2.19-2.262.60.13116261.13198.1
2.26-2.332.60.12816341.177198
2.33-2.412.70.1116491.162198.4
2.41-2.512.70.116291.254198.6
2.51-2.632.60.09416691.304198.5
2.63-2.762.60.08416421.496198.4
2.76-2.942.70.07616401.513198.6
2.94-3.162.60.06916611.638198.9
3.16-3.482.60.05916761.486199.1
3.48-3.992.60.05616631.546198.9
3.99-5.022.60.05316651.53198.6
5.02-502.60.05116821.605196.4

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
MOLREPphasing
REFMAC5.5.0110refinement
PDB_EXTRACT3.11data extraction
ADSCQuantumdata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.85→50 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.934 / Occupancy max: 1 / Occupancy min: 1 / SU B: 3.629 / SU ML: 0.106 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.152 / 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
Rfree0.2452 1631 5.1 %RANDOM
Rwork0.2026 ---
obs0.2048 31872 95.12 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 55.44 Å2 / Biso mean: 22.2837 Å2 / Biso min: 6.84 Å2
Baniso -1Baniso -2Baniso -3
1--2.32 Å20 Å2-0.71 Å2
2--3.67 Å20 Å2
3----1.76 Å2
Refinement stepCycle: LAST / Resolution: 1.85→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2544 0 165 169 2878
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0222792
X-RAY DIFFRACTIONr_bond_other_d0.0040.021823
X-RAY DIFFRACTIONr_angle_refined_deg1.8162.0173832
X-RAY DIFFRACTIONr_angle_other_deg1.1413.0184443
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8675322
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.61324.167120
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.10215407
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.3011515
X-RAY DIFFRACTIONr_chiral_restr0.110.2410
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0213041
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02544
X-RAY DIFFRACTIONr_mcbond_it0.9131.51638
X-RAY DIFFRACTIONr_mcbond_other0.2381.5642
X-RAY DIFFRACTIONr_mcangle_it1.66322655
X-RAY DIFFRACTIONr_scbond_it2.58431154
X-RAY DIFFRACTIONr_scangle_it3.8664.51177
LS refinement shellResolution: 1.853→1.901 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.385 100 -
Rwork0.279 1879 -
all-1979 -
obs--80.61 %

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