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Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA
S Tan1, Y Hunziker, L Pellegrini
1Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, ETH-Zürich, CH-8093, Switzerland.
Abstract:
We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including 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 how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.