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- PDB-2p4t: Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+... -

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Basic information

Entry
Database: PDB / ID: 2p4t
TitleStructure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode
ComponentsDihydrofolate reductase type 2
KeywordsOXIDOREDUCTASE / bacterial infections / folate metabolism / NADP+ / R67 DHFR / Symmetric binding / trimethoprim-resistance
Function / homology
Function and homology information


response to methotrexate / dihydrofolate reductase / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / response to xenobiotic stimulus / response to antibiotic
Similarity search - Function
Dihydrofolate reductase, type II / R67 dihydrofolate reductase / SH3 type barrels. - #60 / Mechanosensitive ion channel MscS, beta-domain superfamily / Electron transport accessory-like domain superfamily / SH3 type barrels. / Roll / Mainly Beta
Similarity search - Domain/homology
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Dihydrofolate reductase type 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.15 Å
AuthorsDivya, N. / Grifith, E. / Narayana, N.
Citation
Journal: Protein Sci. / Year: 2007
Title: Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode.
Authors: Divya, N. / Grifith, E. / Narayana, N.
#1: Journal: Acta Crystallogr.,Sect.D / Year: 2006
Title: High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site
Authors: Narayana, N.
#2: Journal: Nat.Struct.Biol. / Year: 1995
Title: A Plasmid-Encoded Dihydrofolate Reductase from Trimethoprim-Resistant Bacteria has a Novel D2-Symmetric Active Site
Authors: Narayana, N. / Matthews, D.A. / Howell, E.E. / Xuong, N.
#3: Journal: Protein Eng. / Year: 1997
Title: A glutamine 67-->histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition
Authors: Park, H. / Bradrick, T.D. / Howell, E.E.
History
DepositionMar 13, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2007Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
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 type 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)7,4862
Polymers6,7431
Non-polymers7431
Water1,40578
1
A: Dihydrofolate reductase type 2
hetero molecules

A: Dihydrofolate reductase type 2
hetero molecules

A: Dihydrofolate reductase type 2
hetero molecules

A: Dihydrofolate reductase type 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,9448
Polymers26,9704
Non-polymers2,9744
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_665-x+1,-y+1,z1
crystal symmetry operation8_666-y+1,-x+1,-z+11
crystal symmetry operation15_556y,x,-z+11
Unit cell
Length a, b, c (Å)67.460, 67.460, 52.860
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number98
Space group name H-MI4122
DetailsThe biological assembly is comprised of four subunits generated by symmetry axes. The crystallographic 222 symmetry generates the biologically active tetramer. x,y,z; -x+1,-y+1,z; y,x,-z+1; -y+1,-x+1,-z+1

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Components

#1: Protein Dihydrofolate reductase type 2 / Dihydrofolate reductase type II


Mass: 6742.546 Da / Num. of mol.: 1 / Mutation: Residues 1-16 are cleaved and Q67H mutation
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Strain: TMP-RESISTANT, CONTAINING R67 DHFR OVERPRODUCING PLASMID PLZ1
Production host: Escherichia coli (E. coli) / References: UniProt: P00383, dihydrofolate reductase
#2: 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
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 78 / 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: 2.23 Å3/Da / Density % sol: 44.81 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1M Tris-Hcl buffer, 20% PEG1000 and 10% MPD, pH 7.5, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: F1 / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Apr 23, 2005
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 1.15→20 Å / Num. all: 19048 / Num. obs: 18488 / % possible obs: 86.6 % / Observed criterion σ(I): 1 / Redundancy: 7.1 % / Rsym value: 0.039 / Net I/σ(I): 30.2
Reflection shellResolution: 1.15→1.24 Å / Redundancy: 1.4 % / Mean I/σ(I) obs: 2.4 / Num. unique all: 1609 / Rsym value: 0.285 / % possible all: 60.2

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
CNSrefinement
TNT5Erefinement
HKL-2000data reduction
HKL-2000data scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1VIE

1vie


Resolution: 1.15→20 Å / Cross valid method: THROUGHOUT / σ(F): 1 / σ(I): 1 / Stereochemistry target values: Engh & Huber
Details: HOH 149 HAS PARTIAL OCCUPANCY. WHEN COFACTOR IS BOUND, THE HOH 149 IS ABSENT. HOH 146 ALSO HAS PARTIAL OCCUPANCY.THE ADENOSINE PHOSPHATE PORTION OF THE COFACTOR IS NOT SEEN IN THE DENSITY. ...Details: HOH 149 HAS PARTIAL OCCUPANCY. WHEN COFACTOR IS BOUND, THE HOH 149 IS ABSENT. HOH 146 ALSO HAS PARTIAL OCCUPANCY.THE ADENOSINE PHOSPHATE PORTION OF THE COFACTOR IS NOT SEEN IN THE DENSITY. THEREFORE, THE RESPECTIVE ATOMIC COORDINATES ARE MISSING IN THIS LIST.
RfactorNum. reflection% reflectionSelection details
Rfree0.215 --Random
Rwork0.19 ---
all0.195 19048 --
obs0.193 18488 84 %-
Refinement stepCycle: LAST / Resolution: 1.15→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms445 0 26 78 549
LS refinement shellResolution: 1.15→1.24 Å
RfactorNum. reflection% reflection
Rfree0.215 950 -
Rwork0.19 --
obs-18488 84 %

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