Crystallization of the yeast MAT alpha 2/MCM1/DNA ternary complex: Generalmethods and principles for protein/DNA cocrystallization

We describe our efforts to crystallize binary MCM1/DNA and ternary MAT alph a 2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that re sulted in high-resolution crystals of the MAT alpha 2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, incl uding improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of h ow straight and bent DNA with single base-pair overhangs can pack end-to-en d in a crystal. (C) 2000 Academic Press.