3SFM
Novel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structure
Summary for 3SFM
| Entry DOI | 10.2210/pdb3sfm/pdb |
| Descriptor | Dihydrofolate reductase type 2, (4R)-2-METHYLPENTANE-2,4-DIOL (3 entities in total) |
| Functional Keywords | oxidoreductase, dihydrofolate reductase, antibiotic resistance, in situ proteolysis, type ii dhfr, sh3, reductase, dhf and nadph-binding |
| Biological source | Escherichia coli |
| Total number of polymer chains | 1 |
| Total formula weight | 7088.03 |
| Authors | Yachnin, B.J.,Berghuis, A.M. (deposition date: 2011-06-13, release date: 2011-11-02, Last modification date: 2023-09-13) |
| Primary citation | Yachnin, B.J.,Colin, D.Y.,Volpato, J.P.,Ebert, M.,Pelletier, J.N.,Berghuis, A.M. Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure. Acta Crystallogr.,Sect.F, 67:1316-1322, 2011 Cited by PubMed Abstract: Trimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF. Using a modified crystallization protocol for this enzyme that included in situ proteolysis using chymotrypsin, the tandem dimer was crystallized and the structure was solved at 1.4 Å resolution. Surprisingly, only wild-type protomers were incorporated into the crystal. Further experiments demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymotrypsin cleavage site had been introduced by these mutations. PubMed: 22102224DOI: 10.1107/S1744309111030417 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.4 Å) |
Structure validation
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