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- PDB-6cxk: E. coli DHFR substrate complex with Dihydrofolate -

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Basic information

Entry
Database: PDB / ID: 6cxk
TitleE. coli DHFR substrate complex with Dihydrofolate
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / DHFR / dihydrofolate reductase / dihydrofolate / complex
Function / homology
Function and homology information


dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / cytosol
Similarity search - Function
Dihydrofolate reductase / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
DIHYDROFOLIC ACID / Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli O6:H1 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.108 Å
AuthorsCao, H. / Rodrigues, J. / Benach, J. / Frommelt, A. / Morisco, L. / Koss, J. / Shakhnovich, E. / Skolnick, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118039 United States
CitationJournal: Commun Biol / Year: 2018
Title: The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release.
Authors: Cao, H. / Gao, M. / Zhou, H. / Skolnick, J.
History
DepositionApr 3, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 9, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Oct 4, 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 / pdbx_struct_conn_angle / struct_conn
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_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.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_symmetry / _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.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,52211
Polymers18,8481
Non-polymers67310
Water3,639202
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)33.698, 51.525, 77.421
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Dihydrofolate reductase


Mass: 18848.223 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O6:H1 (strain CFT073 / ATCC 700928 / UPEC) (bacteria)
Strain: CFT073 / ATCC 700928 / UPEC / Gene: folA, c0058 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABQ5, dihydrofolate reductase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-DHF / DIHYDROFOLIC ACID


Mass: 443.413 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H21N7O6 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 202 / 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: 1.78 Å3/Da / Density % sol: 31.02 %
Description: Rectangular blocks with longest dimension 0.2-0.7 mm appeared in 2-5 days.
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Sitting drop of final volume of 0.8 uL was set up by 1:1 v/v mixing of 15 mg/mL E. coli DHFR working stock with reservoir solution containing 0.1M MES pH 6.5, 27.5% PEG 3350, 0.4M MgCl2. E. ...Details: Sitting drop of final volume of 0.8 uL was set up by 1:1 v/v mixing of 15 mg/mL E. coli DHFR working stock with reservoir solution containing 0.1M MES pH 6.5, 27.5% PEG 3350, 0.4M MgCl2. E. coli DHFR working stock was prepared from a 1:1 v/v mixture of 30 mg/ml protein in 20 mM Tris pH 8, 1mM DTT with 50 mM HEPES pH 7.3, 100 mM NaCl. Crystals harvested after 2 weeks growth and cryoprotected with mitegen LV oil before flash freezing in N2.
Temp details: Room temperature 20-22 degrees celcius

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 31-ID / Wavelength: 0.97931 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Nov 30, 2017 / Details: Diamond(111)
RadiationMonochromator: Kohzu HLD-4 Double Crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97931 Å / Relative weight: 1
ReflectionResolution: 1.108→30.949 Å / Num. obs: 53265 / % possible obs: 97.9 % / Redundancy: 6.255 % / Biso Wilson estimate: 11.47 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.067 / Rrim(I) all: 0.073 / Χ2: 1.003 / Net I/σ(I): 13.44
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.108-1.182.8040.8611.1675790.5711.05187.3
1.18-1.265.510.7822.2981650.8180.864100
1.26-1.367.0640.5873.6576170.9190.633100
1.36-1.497.1340.3855.5270210.9590.415100
1.49-1.667.1780.20310.3463970.9870.218100
1.66-1.927.2050.10518.4656580.9960.113100
1.92-2.357.1730.05632.4948400.9980.061100
2.35-3.327.1030.03845.0637910.9990.04199.9
3.32-30.9496.6930.02456.1421970.9990.02699.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PHENIX1.8.4_1496refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7DFR

7dfr


Resolution: 1.108→30.949 Å / SU ML: 0.14 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 23
RfactorNum. reflection% reflection
Rfree0.1961 1993 3.75 %
Rwork0.1786 --
obs0.1792 53202 97.82 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 72.59 Å2 / Biso mean: 18.63 Å2 / Biso min: 7.68 Å2
Refinement stepCycle: final / Resolution: 1.108→30.949 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1328 0 60 202 1590
Biso mean--27.1 28.6 -
Num. residues----165
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0091476
X-RAY DIFFRACTIONf_angle_d1.3342018
X-RAY DIFFRACTIONf_chiral_restr0.078205
X-RAY DIFFRACTIONf_plane_restr0.007268
X-RAY DIFFRACTIONf_dihedral_angle_d13.574540
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.108-1.13570.42211080.40292778288675
1.1357-1.16640.35581370.33063497363495
1.1664-1.20070.29451420.296636643806100
1.2007-1.23950.30411440.257736843828100
1.2395-1.28380.23851440.23937093853100
1.2838-1.33520.22061450.222637143859100
1.3352-1.39590.23311430.21636843827100
1.3959-1.46950.22851460.190437313877100
1.4695-1.56160.18061430.180237023845100
1.5616-1.68220.17591460.16537443890100
1.6822-1.85140.17561460.161837483894100
1.8514-2.11930.17411480.148137873935100
2.1193-2.66980.19131480.154138073955100
2.6698-30.96120.16541530.155839604113100

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