Protocol, cooling

The complementary strands are mixed in 1 1 ratio and annealed by heating to 80 C followed by slow cooling (Protocol 15.2). The annealed double-stranded oligomer can be further purified by ion-exchange chromatography (Protocol 15.3) to remove single strands. Protocols 15.1 and 15.3 are modified from the methods described by Aggarwal (1990) and Joachimiak et al. (1987). 

Briinger, A. T., Krukowski, A., and Erickson, J. W. Slow-cooling protocols for crystallographic refinement by simulated annealing. Acta Cryst. A46, 585-593 (1990). 

Constant temperature torsion angle refinements (Fig. 1) outperform the slow-cooling protocol on average, dramatically so if one only considers the best model from each series. The torsion angle refinements are able to correct backbone atom r.m.s. coordinate deviations of at least 1.65 A. Clearly, the backbone atom r.m.s. coordinate deviation is only available if one knows the answer in advance. Fig. 2, however, shows the strong correlation between Rfree and backbone r.m.s. coordinate deviations. Thus in practice Rfree Can be used to consistently identify the best models from a series of refinements. 

Boolean variables (p. 382) Car-ParrineUo algorithm (p. 377) cellular automaton (p. 381) characteristic frequency (p. 359) cooling protocol (p. 370) entropy (p. 353) force field (p. 345) free energy (p. 353) global minimum (p. 352) global optimization (p. 354)... 

GUAN M, RAWSON D M and ZHANG T (2008) Cryopreservation of zebrafish Danio rerio) oocytes using improved controlled slow cooling protocols. Cryobiology, 56, 204-208. 

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