PDB-2rh2: High Resolution DHFR R-67

Basic information

• Entry: Database: PDB / ID: 2rh2
• Title: High Resolution DHFR R-67
• Components: Dihydrofolate reductase type 2
• Keywords: OXIDOREDUCTASE / FOLATE METABOLISM / PLASMID-ENCODED R67 DHFR / TMP-RESISTANT DHFR / Antibiotic resistance / Methotrexate resistance / NADP / One-carbon metabolism / 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:

Dihydrofolate reductase type 2

• Biological species: Escherichia coli (E. coli)
• Method: X-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 0.96 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 5, 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

hetero molecules

Summary:

• 7.21 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	7,205	5
Polymers	6,733	1
Non-polymers	473	4
Water	2,396	133

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&software
• 28.8 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	28,821	20
Polymers	26,930	4
Non-polymers	1,891	16
Water	72	4

Symmetry operations:

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

Calculated values:

Buried area	5720 Å2
Method	PISA

2

A: Dihydrofolate reductase type 2

hetero molecules

A: Dihydrofolate reductase type 2

hetero molecules

Summary:

• defined by software
• 14.4 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	14,410	10
Polymers	13,465	2
Non-polymers	945	8
Water	36	2

Symmetry operations:

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

Calculated values:

Buried area	1810 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	67.810, 67.810, 51.970
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 by the operations: x, y, z; y, x, -z; -x, -y, z; -y, -x, z

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 genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Description: TMP-RESISTANT, CONTAINING R67 DHFR OVERPRODUCING PLASMID PLZ1
Production host: Escherichia coli (E. coli) / References: UniProt: P00383, dihydrofolate reductase

#2: Chemical

A-400 A-401 A-402 A-403
ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL

Details:

Mass: 118.174 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₆H₁₄O₂ / Comment: precipitant*YM

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 133 / 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: 2.22 ų/Da / Density % sol: 44.55 %
• Crystal grow: Temperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8 ; Details: 50% 2-methyl-2,4-pentanediol (MPD), 100 mM Tris at pH 8.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
• Detector: Type: RIGAKU SATURN 92 / Detector: CCD / Date: Apr 15, 2007 / Details: Osmic VariMax HF confocal mirrors
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1.5418 Å / Relative weight: 1
• Reflection: Resolution: 0.96→25 Å / Num. all: 32858 / Num. obs: 32858 / % possible obs: 96.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 16.3 % / R_merge(I) obs: 0.1 / R_sym value: 0.1 / Net I/σ(I): 27.1
• Reflection shell: Resolution: 0.96→0.99 Å / Redundancy: 6.7 % / R_merge(I) obs: 0.657 / Mean I/σ(I) obs: 3.6 / Num. unique all: 3331 / R_sym value: 0.657 / % possible all: 91.1

Processing

Software

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

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: 1VIE

1vie

Resolution: 0.96→25 Å / Cor.coef. F_o:F_c: 0.983 / Cor.coef. F_o:F_c free: 0.985 / SU B: 0.383 / SU ML: 0.01 / Isotropic thermal model: anisotropic / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.017 / ESU R Free: 0.017 / Stereochemistry target values: Engh & Huber / Details: All protein hydrogens are included.

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.11683	1776	5.1 %	RANDOM
Rwork	0.10316	-	-	-
all	0.10387	32858	-	-
obs	0.10387	32858	93.28 %	-

• Solvent computation: Ion probe radii: 1 Å / Shrinkage radii: 1 Å / VDW probe radii: 1.4 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 10.397 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	0.4 Å2	0 Å2	0 Å2
2-	-	0.4 Å2	0 Å2
3-	-	-	-0.8 Å2

Refinement step

Cycle: LAST / Resolution: 0.96→25 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1082	0	28	133	1243

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.016	0.021	607
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.02	527
X-RAY DIFFRACTION	r_angle_refined_deg	1.648	1.958	846
X-RAY DIFFRACTION	r_angle_other_deg	0.742	3	1209
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.29	5	80
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	36.053	23.704	27
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	11.853	15	86
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	9.956	15	4
X-RAY DIFFRACTION	r_chiral_restr	0.101	0.2	83
X-RAY DIFFRACTION	r_gen_planes_refined	0.01	0.02	725
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	149
X-RAY DIFFRACTION	r_nbd_refined	0.377	9.999	128
X-RAY DIFFRACTION	r_nbd_other	0.257	9.999	581
X-RAY DIFFRACTION	r_nbtor_refined	0.207	0.5	278
X-RAY DIFFRACTION	r_nbtor_other	0.097	0.5	355
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.224	9.999	127
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	0.521	9.999	19
X-RAY DIFFRACTION	r_symmetry_vdw_other	0.344	9.999	54
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.2	9.999	40
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	2.343	2.5	368
X-RAY DIFFRACTION	r_mcbond_other	2.353	2.5	142
X-RAY DIFFRACTION	r_mcangle_it	3.002	4	592
X-RAY DIFFRACTION	r_scbond_it	3.467	3	284
X-RAY DIFFRACTION	r_scangle_it	4.648	5	254
X-RAY DIFFRACTION	r_rigid_bond_restr	2.002	2	628
X-RAY DIFFRACTION	r_sphericity_free	15.827	5	114
X-RAY DIFFRACTION	r_sphericity_bonded	6.735	5	555

LS refinement shell

Resolution: 0.96→0.985 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.248	106	-
Rwork	0.233	2052	-
obs	-	-	80.01 %