Crystallization, dehydration and preliminary X-ray analysis of excisionase (Xis) proteins cooperatively bound to DNA

Abstract

This paper describes the crystallization, dehydration and preliminary X-ray data analysis of a complex containing several bacteriophage lambda excisionase (Xis) [Bushman et al. (1984). Cell, 39, 699-706] proteins cooperatively bound to a 33-mer DNA duplex (Xis-DNA(X1-X2)). Xis is expected to recognize this regulatory element in a novel manner by cooperatively binding and distorting multiple head-to-tail orientated DNA-binding sites. Crystals of this complex belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 107.7, c = 73.5 angstroms, alpha = beta = 90, gamma = 120 degrees. Based on the unit-cell parameters for the asymmetric unit, V(M) is 3.0 A(3) Da(-1), which corresponds to a solvent content of approximately 59%. The approaches used to crystallize the unusually long DNA fragment in the complex and the dehydration technique applied that dramatically improved the diffraction of the crystals from 10 to 2.6 angstroms are discussed.

Inducing phase changes in crystals of macromolecules: Status and perspectives for controlled crystal dehydration

“…From the earliest days of macromolecular crystallography (MX) the effects and potential benefits of crystal dehydration have been recognised (Berthou et al, 1972;Einstein and Low, 1962;Huxley and Kendrew, 1953;Perutz, 1946). However, the lack of a method to accurately control and change the RH of the gas surrounding a crystal and, crucially, characterise changes in diffraction quality during dehydration, has limited the use of this technique to a few cases, with improvement in diffraction often discovered by chance (Abergel, 2004;Adachi et al, 2009;Cramer et al, 2000;Esnouf et al, 1998;Fratini et al, 1982;Gupta et al, 2010;Heras et al, 2003;Kuo et al, 2003;Nakamura et al, 2007;Sam et al, 2006;Vijayalakshmi et al, 2008;Yap et al, 2007). Many techniques have been developed to dehydrate crystals (Amunts et al, 2007;Heras and Martin, 2005;Newman, 2006) but as these often involve the transfer of crystals between solutions, they can prove difficult to reproduce.…”

Automation and Experience of Controlled Crystal Dehydration: Results from the European Synchrotron HC1 Collaboration

“…This technique has been known to alter the properties of crystals since Max Perutz’s pioneering experiments in the 1940s . Since then, many examples have been found, but these occurred often by accident − rather than design. − In an effort to characterize and control these effects, several devices have been developed in order to control the humidity surrounding crystals. − The more recently developed devices have been designed with the goal of being compatible with X-ray sources in order to directly observe the effects of humidity on the diffraction characteristics of the crystal being studied. − These devices moved the study of crystal dehydration into a new realm as humidity could be controlled precisely while monitoring the effects through diffraction, but they were often difficult to install on rotating anode generators or synchrotron beamlines. The latter limitation stimulated the development of a new device at the EMBL Grenoble Outstation, the HC1b. , The HC1b produces an air stream with controlled relative humidity using a dispensing nozzle, in the same manner as cryostream devices produce a nitrogen flow at 100 K, and is therefore easy to integrate with most diffractometers.…”

Unit-cell response of tetragonal hen egg white lysozyme upon controlled relative humidity variation

“…The direct association between dehydration phenomena, unit-cell parameter variation, observation of new tertiary structures and improvement in data quality at lower humidity levels has been recognized since Max Perutz's revolutionary experiments in the 1940s (Perutz, 1946 been a few studies reporting major changes in unit-cell dimensions upon rH variation (Huxley & Kendrew, 1953;Einstein & Low, 1962;Perutz, 1964;Salunke et al, 1985), while the relation between dehydration and enhancement of diffraction quality has been exploited in several projects either accidentally (Esnouf et al, 1998;Kuo et al, 2003;Sam et al, 2006) or deliberately (Henrich et al, 2003;Kyrieleis et al, 2005), leading to the production of several devices over the years (Kiefersauer et al, 2000).…”