Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus:Purification, crystallization and structure determination

We describe the crystallization and structure determination of the 30 S rib osomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 Angstrom resolution were used as a starting point to impr ove the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved l ess important than attention to detail in yielding crystals that diffracted beyond 3 Angstrom resolution. Despite improvements in technology and metho dology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of t he asymmetric unit, crystal variability and sensitivity to radiation damage . Some steps that were useful for determination of the atomic structure wer e: the use of anomalous scattering from the LIII edges of osmium and luteti um to obtain the necessary phasing signal; the use of tunable, third-genera tion synchrotron sources to obtain data of reasonable quality at high resol ution; collection of derivative data precisely about a mirror plane to pres erve small anomalous differences between Bijvoet mates despite extensive ra diation damage and multicrystal scaling; the pre-screening of crystals to e nsure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; preincubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction w ith biochemical data on protein-RNA interactions, to map out the architectu re of the 30 S subunit prior to the construction of a detailed atomic-resol ution model. (C) 2001 Academic Press.