In situX-ray data collection from highly sensitive crystals ofPseudomonas putidaPtxS in complex with DNA

Abstract

Pseudomonas putidaPtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons,kguandgad. PtxS controls the expression of thekguandgadoperons as well as its own transcription. The PtxS operator is a perfect palindrome, 5′-TGAAACCGGTTTCA-3′, which is present in all three promoters. Crystallization of native PtxS failed, and PtxS–DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10 × 10 µm. The crystal belonged to the trigonal space groupP3, with unit-cell parametersa=b= 213.71,c = 71.57 Å. Only unhandled crystals grown in capillaries of 0.1 mm inner diameter diffracted X-rays to 1.92 Å resolution.