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- PDB-3sfm: Novel crystallization conditions for tandem variant R67 DHFR yiel... -

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Basic information

Entry
Database: PDB / ID: 3sfm
TitleNovel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structure
ComponentsDihydrofolate reductase type 2
KeywordsOXIDOREDUCTASE / dihydrofolate reductase / antibiotic resistance / in situ proteolysis / type II DHFR / SH3 / reductase / DHF and NADPH-binding
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
Dihydrofolate reductase type 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsYachnin, B.J. / Berghuis, A.M.
CitationJournal: Acta Crystallogr.,Sect.F / Year: 2011
Title: Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure.
Authors: Yachnin, B.J. / Colin, D.Y. / Volpato, J.P. / Ebert, M. / Pelletier, J.N. / Berghuis, A.M.
History
DepositionJun 13, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2011Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2011Group: Database references
Revision 1.2Sep 13, 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 / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrofolate reductase type 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)7,0884
Polymers6,7341
Non-polymers3553
Water1,06359
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)28,35216
Polymers26,9344
Non-polymers1,41812
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_555-y,-x,-z1
crystal symmetry operation10_555-x,-y,z1
crystal symmetry operation15_555y,x,-z1
Buried area6800 Å2
ΔGint-29 kcal/mol
Surface area11700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.691, 67.691, 51.760
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number98
Space group name H-MI4122

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Components

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


Mass: 6733.513 Da / Num. of mol.: 1 / Fragment: Residues 21-78
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: dfrB / Plasmid: pQE-32-derived / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(pRep4) / References: UniProt: P00383, dihydrofolate reductase
#2: Chemical ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL


Mass: 118.174 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 59 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHE FULL LENGTH OF THE EXPRESSED PROTEIN (TOTAL OF 171 residues: ...THE FULL LENGTH OF THE EXPRESSED PROTEIN (TOTAL OF 171 residues: MRGSHHHHHHGIHMERSSNEVSNPVAGNFVFPSDATFGMGDRVRKKSGAAWQGQIVGWYCTNLTPEGYAVESEAHPGSINSFPVAALERINELMERSSNEVSNPVAGNFVFPSDATFGMGDRVRKKSGAAWQGQIVGWYCTNLTPEGYAVESEAHPGSVQIYPVAALERIN) WAS CRYSTALLIZED USING IN SITU PROTEOLYSIS WITH CHYMOTRYPSIN. THIS PROCESS INVOLVES THE DEGRADATION OF THE N-TERMINAL 109 RESIDUES OF THE PROTEIN, LEAVING ONLY THE C-TERMINAL 62 RESIDUES WHICH CORRESPOND TO THE DATE BASE REFERENCE SEQUENCE RESIDUE NUMBERING OF 17-78.

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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: 2.35 Å3/Da / Density % sol: 47.7 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.2
Details: 0.1 M sodium phosphate, 55% MPD, pH 7.2, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 93 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Sep 16, 2009 / Details: Osmic Confocal Blue
RadiationMonochromator: Rotating copper anode (1.5418) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.4→50 Å / Num. all: 12169 / Num. obs: 12169 / % possible obs: 100 % / Redundancy: 23.8 % / Biso Wilson estimate: 16.788 Å2 / Rmerge(I) obs: 0.061 / Rsym value: 0.061 / Net I/σ(I): 18.5
Reflection shellResolution: 1.4→1.43 Å / Redundancy: 13.9 % / Rmerge(I) obs: 0.371 / Num. unique all: 802 / Rsym value: 0.371 / % possible all: 100

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Processing

Software
NameClassification
StructureStudiodata collection
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB Entry 2RH2

2rh2


Resolution: 1.4→23.97 Å / Cor.coef. Fo:Fc: 0.971 / Cor.coef. Fo:Fc free: 0.979 / SU B: 1.218 / SU ML: 0.023 / Isotropic thermal model: Anisotropic / Cross valid method: THROUGHOUT / ESU R Free: 0.052 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.16387 626 5.1 %RANDOM
Rwork0.14445 ---
all0.14548 11533 --
obs0.14548 11533 99.91 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 17.816 Å2
Baniso -1Baniso -2Baniso -3
1-0.07 Å20 Å20 Å2
2--0.07 Å20 Å2
3----0.14 Å2
Refinement stepCycle: LAST / Resolution: 1.4→23.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms443 0 24 59 526
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0210.022507
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.9171.948694
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.923559
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.84923.33324
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.8131576
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.046154
X-RAY DIFFRACTIONr_chiral_restr0.1350.276
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.021379
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.2981.5292
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it3.4122472
X-RAY DIFFRACTIONr_scbond_it4.8723215
X-RAY DIFFRACTIONr_scangle_it6.924.5221
X-RAY DIFFRACTIONr_rigid_bond_restr2.5133507
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.4→1.435 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.565 42 -
Rwork0.525 826 -
obs-868 98.75 %

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