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J. Biol. Chem., Vol. 264, Issue 12, 6737-6740, 04, 1989

Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor

L Betts, L Frick, R Wolfenden and CW Carter Jr
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260.

We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x- ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5- fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.
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