PDB-2rk1: DHFR R67 Complexed with NADP and dihydrofolate

Basic information

• Entry: Database: PDB / ID: 2rk1
• Title: DHFR R67 Complexed with NADP and dihydrofolate
• Components: Dihydrofolate reductase type 2
• Keywords: OXIDOREDUCTASE / NADP / dihydrofolate / asymmetric ligand binding / Antibiotic resistance / Methotrexate resistance / One-carbon metabolism / Plasmid / Trimethoprim resistance

Function / homology

Function:

response to methotrexate / dihydrofolate reductase / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / response to xenobiotic stimulus / response to antibiotic

Domain/homology:

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

Component:

DIHYDROFOLIC ACID / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Dihydrofolate reductase type 2

• Biological species: Escherichia coli (E. coli)
• Method: X-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.26 Å
• Authors: Krahn, J.M. / London, R.E.
• Citation: Journal: Biochemistry / Year: 2007; Title: Crystal structure of a type II dihydrofolate reductase catalytic ternary complex.; Authors: Krahn, J.M. / Jackson, M.R. / DeRose, E.F. / Howell, E.E. / London, R.E.

History

Deposition	Oct 16, 2007	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jun 3, 2008	Provider: repository / Type: Initial release
Revision 1.1	Jul 13, 2011	Group: Version format compliance
Revision 1.2	Aug 30, 2023	Group: Data collection / Database references / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

Assembly

Deposited unit

A: Dihydrofolate reductase type 2

A: DIHYDROFOLIC ACID

hetero molecules

Summary:

• 8.27 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	8,274	6
Polymers	6,733	1
Non-polymers	1,541	5
Water	1,964	109

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

Summary:

• defined by author
• 33.1 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	33,095	24
Polymers	26,930	4
Non-polymers	6,165	20
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	8_555	-y,-x,-z	1
crystal symmetry operation	10_555	-x,-y,z	1
crystal symmetry operation	15_555	y,x,-z	1

Unit cell

Length a, b, c (Å)	67.804, 67.804, 52.201
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	98
Space group name H-M	I4122

• Details: The biological assembly is a tetramer generated from the monomer in the asymmetric unit with the symmetry operations: x, y, z; y, x, -z; -x, -y, z; -y, -x, -z; The NADP and dihydrofolate ligands should be included only for the identity operator, and assigned full occupancy. Water molecules that clash with NADP or dihydrofolate should then be removed to obtain the set of waters that are valid for the tetramer. Tyr69 and Gln67 are also asymmetric, but the correlation of their alternate conformations to the asymmetric ligand binding has not been conclusively determined.

Components

#1: Protein

A Dihydrofolate reductase type 2 / Dihydrofolate reductase type II

Details:

Mass: 6732.528 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
Strain: TMP-RESISTANT, CONTAINING R67 DHFR OVERPRODUCING PLASMID PLZ1
References: UniProt: P00383, dihydrofolate reductase

#2: Chemical

A A-0 ChemComp-DHF / DIHYDROFOLIC ACID / Dihydrofolic acid

Details:

Mass: 443.413 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₁₉H₂₁N₇O₆

#3: Chemical

A-1 ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate

Details:

Mass: 743.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂₁H₂₈N₇O₁₇P₃

#4: Chemical

A-500 A-501 A-502 ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL / 2-Methyl-2,4-pentanediol

Details:

Mass: 118.174 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C₆H₁₄O₂ / Comment: precipitant*YM

#5: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 109 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 2.23 ų/Da / Density % sol: 44.79 %
• Crystal grow: Temperature: 277 K / pH: 8 ; Details: 50% 2-methyl-2,4-pentanediol (MPD), 100 mM Tris, pH 8.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K, pH 8.00

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418
• Detector: Type: RIGAKU SATURN 92 / Detector: CCD / Date: Mar 17, 2005 / Details: VARIMAX HF, CONFOCAL MIRRORS
• Radiation: Monochromator: CONFOCAL MIRRORS (VARIMAX HF) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1.5418 Å / Relative weight: 1
• Reflection: Resolution: 1.26→50 Å / Num. obs: 15838 / % possible obs: 99.6 % / Observed criterion σ(I): -3 / Redundancy: 12.4 % / R_merge(I) obs: 0.072 / Net I/σ(I): 14.1
• Reflection shell: Resolution: 1.26→1.29 Å / Redundancy: 5.4 % / R_merge(I) obs: 0.499 / Mean I/σ(I) obs: 3 / % possible all: 96.5

Processing

Software

Name	Version	Classification
REFMAC	5.2.0019	refinement
HKL-2000		data reduction
HKL-2000		data scaling
CNS		phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: 2RH2

2rh2

Resolution: 1.26→26.22 Å / Cor.coef. F_o:F_c: 0.974 / Cor.coef. F_o:F_c free: 0.967 / SU B: 1.057 / SU ML: 0.021 / Isotropic thermal model: Anisotropic / Cross valid method: THROUGHOUT / ESU R: 0.045 / ESU R Free: 0.042 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.15231	854	5.1 %	RANDOM, preserved from 2RH2
Rwork	0.12266	-	-	-
obs	0.12419	15838	99.67 %	-
all	-	16692	-	-

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 14.403 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	0 Å2	0 Å2	0 Å2
2-	-	0 Å2	0 Å2
3-	-	-	0 Å2

Refinement step

Cycle: LAST / Resolution: 1.26→26.22 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1034	0	85	122	1241

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.016	0.021	649
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.02	497
X-RAY DIFFRACTION	r_angle_refined_deg	2.121	2.08	913
X-RAY DIFFRACTION	r_angle_other_deg	0.87	3	1139
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.219	5	78
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	34.012	24.231	26
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	12.569	15	83
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	11.459	15	3
X-RAY DIFFRACTION	r_chiral_restr	0.102	0.2	89
X-RAY DIFFRACTION	r_gen_planes_refined	0.009	0.02	740
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	139
X-RAY DIFFRACTION	r_nbd_refined	0.298		121
X-RAY DIFFRACTION	r_nbd_other	0.245		520
X-RAY DIFFRACTION	r_nbtor_refined	0.225	0.5	311
X-RAY DIFFRACTION	r_nbtor_other	0.106	0.5	387
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.293	99.999	113
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined	0.163		17
X-RAY DIFFRACTION	r_symmetry_vdw_other	0.326		72
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.141	99.999	35
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	2.499	2	364
X-RAY DIFFRACTION	r_mcbond_other	1.359	2	137
X-RAY DIFFRACTION	r_mcangle_it	3.166	3	575
X-RAY DIFFRACTION	r_scbond_it	4.852	5	371
X-RAY DIFFRACTION	r_scangle_it	6.56	10	338
X-RAY DIFFRACTION	r_rigid_bond_restr	2.563	3	714
X-RAY DIFFRACTION	r_sphericity_free	20.897	8	122
X-RAY DIFFRACTION	r_sphericity_bonded	6.865	8	598

LS refinement shell

Resolution: 1.26→1.293 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.376	54	-
Rwork	0.306	1096	-
obs	-	-	95.36 %