AH

Crystallization is the primary rate-limiting step in protein structure determination. It has been our expe-rience over approximately 10 years that crystals are obtained in about 20 % of the proteins attempted and that only about 10 % of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization ‘of mi-croheterogeneity in a protein regarded as pure by con-ventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of pre-cipitants, crystallization conditions, and additives failed to discover conditions from which usable crys-tals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution. Creatine kinase (EC 2.7.3.2, ATP:creatine N-phosphotrans-ferase) catalyzes the transfer of a phosphate group from phosphocreatine to ADP to regenerate ATP in the following reaction