Figure 1.
Analysis of purified ParC55 and its cysteine mutants by 10% SDS-polyacrylamide gel electrophoresis.
Lane 1, molecular-weight markers; lane 2, wild-type; lane 3, Cys109; Lane 4, Cys110; Lane 5, Cys190; lane 6, Cys294; lane 7, Cys387; lane 8, Cys426; lane 9, Cys437. The molecular weights of the markers are indicated.
Figure 2.
(A) Orthorhombic crystal of Cys426 grown from 8% PEG 20,000, 200 mM sodium chloride, 100 mM Tris-HCl, pH 5.0, 0.1% sodium azide. (B) Hexagonal crystal of ParC55 grown from 8% PEG 20,000, 200 mM sodium chloride, 100 mM Tris-HCl, pH 7.0, 0.1% sodium azide.
Figure 3.
(A) Wild type ParC55 twinned crystal grown in a hanging drop containing 6 µl of the protein solution at 3 mg/ml concentration and 6 µl of the reservoir solution containing 200 mM sodium chloride, 0.09 mM sodium azide, 5% (v/v) ethanol, 5% (v/v) isopropanol, 90 mM Tris-HCl, pH 5.5. The diffraction image was taken from the highest resolution (2.7Å) data set collected at ESRF BM 30A. Crystal to detector distance was 340 mm and the exposure time 160 seconds. (B) Mutant Cys426 crystal grown from 6% PEG 400, 1 mM β-mercaptoethanol, 0.05% sodium azide, 200 mM sodium chloride, 100 mM Tris-HCl, pH 6.5. The image was taken from the data set collected at Rigaku Americas Corporation (The Woodlands, Texas, USA) with crystal to detector distance of 336 mm and an exposure time of 5 min.
Table 1.
Crystals tested for diffraction in-house.
Table 2.
Mutant ParC55 crystals tested for diffraction in-house.
Figure 4.
Orthogonal views of the ParC55 biological dimer from Streptococcus pneumoniae.
(A) Cartoon representation. The ‘towers’ and the CAP-like domains are shown in ice blue; the ‘tails’ along with adjacent helices α14, α18 and α19 are in ochre; the helix α4 in red; the helix α3 in cyan; the 100–122 loop in yellow. The active-site tyrosines are shown in green. Residues responsible for drug-resistance upon mutation are in purple. (B) Electrostatic surface representation. The negatively charged regions are in red and positively charged regions are in blue. Panels were rendered using VMD [27], Pov-Ray and PyMOL [28].