Crystallization and preliminary crystallographic analysis of the site-specific DNA nickase Nb.BspD6I

Abstract

Crystals of site-speci®c DNA nickase Nb.BspD6I (of molecular weight 70.8 kDa) have been grown at 291 K using PEG 8000 as precipitant. The diffraction pattern of the crystal extends to 3.3 A Ê resolution at 100 K. The crystal belongs to space group P2 1 , with unit-cell parameters a = 57.76, b = 90.67, c = 71.71, = 110.1. There is one molecule in the asymmetric unit and the solvent content is estimated to be 53% by volume.

Structural Analysis of the Heterodimeric Type IIS Restriction Endonuclease R.BspD6I Acting as a Complex between a Monomeric Site-specific Nickase and a Catalytic Subunit

“…The purification and crystallization procedures were as described. 18,19 Crystals of the full-length monomeric were grown in space group P2 1 (cell parameters a = 59.5 Å, b =92.6 Å, c =76.3 Å, β = 111.8°) with one molecule per asymmetric unit. X-ray diffraction data (Table 2) were collected on the MPG beamline BW6, DESY/HASYLAB, Hamburg, using a 165 mm MarCCD detector.…”

“…of 3.4 Å (between 96 main-chain atoms). The largest deviation is visible in a segment that forms a loop in ss.BspD6I (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The corresponding segment in Nt.BspD6I (393-414) is more extended and includes a helical stretch (397-407).…”

“…In earlier work, we characterized and crystallized the subunits Nt.BspD6I 17,18 and ss.BspD6I. 19 Protein sequence alignment between ss.BspD6I and the Cterminal domain of Nt.BspD6I revealed 27% identity and 20% similarity.…”

Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

“…Crystal structure analysis of Nt.BspD6I protein was per formed to explain its oligomerization [12] (PDB code 2ewf). Distribution of the charged amino acid residues on the protein surface was investigated.…”

“…It was shown that Nt.BstNBI was not able to form a dimer at low concentrations either in the free state or interacting with the specific DNA [11]. Previously, the conditions for Nt.BspD6I mono mer crystallization without DNA were found, and the crystal structure was solved [12]. However, the forma tion of higher molecular weight complexes of BspD6I with DNA, as compared to the monomer, were observed at the high protein concentrations required for crystallization experiments.…”