High crystallizability under air-exclusion conditions of the full-length LysR-type transcriptional regulator TsaR from Comamonas testosteroni T-2 and data-set analysis for a MIRAS structure-solution approach

Abstract

The full-length LysR-type transcriptional regulator TsaR from Comamonas testosteroni T-2 was heterologously overexpressed in Escherichia coli, purified and stabilized under conditions that favoured its rapid crystallization using the microbatch-under-oil technique. The purified protein was highly crystallizable and two different crystal forms were readily obtained. However, only monoclinic crystals gave diffraction beyond 2 A and there was a slight variation in unit-cell parameters between crystals. The only other LysR-type regulator for which a full-length crystal form is available is CbnR, but no solution could be obtained when this was used as a model in molecular replacement. Mercury and xenon derivatives were therefore produced in order to phase the structure using a MIRAS approach.

Figure 1

(a) Initial crystals of TsaR obtained by hanging-drop vapour diffusion in 1 M glycine pH 8.0 and 0.5 M NaCl. (b) Optimization of the same conditions by the addition of 0.1 M NaI.

Figure 2

Crystals obtained using the microbatch-under-oil technique at 293 K with condition No. 34 of Crystal Screen I (CSI; Hampton Research). (b) The same condition in a 0.2 mm internal diameter capillary. (c) and (d) Crystals obtained using the microbatch-under-oil technique at 293 K with (c) condition No. 20 and (d) condition No. 48 of CSI.

Figure 3

Diffraction patterns of (a) C2 crystals grown using condition No. 34 of CSI and (b) P4₁2₁2 or P4₃2₁2 crystals grown using condition No. 48 of CSI.

Figure 4

Stereographic projection for a data set collected from a crystal grown using condition No. 48 of CSI (P4₁2₁2 or P4₃2₁2); the κ = 180° section shows two peaks of equal height (90% of the origin peak) at polar angles ω = 90, ϕ = 31° and ω = 90, ϕ = 14°. This figure was prepared using XPREP (Sheldrick, 1997 ▶).