THE SUITABILITY OF A MONOFUNCTIONAL REAGENT OF AN UNDECAGOLD CLUSTER FOR PHASING DATA COLLECTED FROM THE LARGE RIBOSOMAL-SUBUNITS FROM BACILLUS-STEAROTHERMOPHILUS

An electron density map of the large ribosomal subunit from Bacillus s tearothermophilus was obtained at 26 Angstrom resolution by single iso morphous replacement (SIR) from a derivative formed by specific quanti tative labeling with a dense undecagold cluster. For derivatization, a mono-functional reagent of this cluster was bound to a sulfhydryl gro up of a purified ribosomal protein, which was in turn reconstituted wi th core particles of a mutant tacking this protein. The native, mutate d, and derivatized 50S ribosomal subunits crystallize under the same c onditions in the same space group. Under favorable conditions, crystal s of the derivatized subunit proved to be isomorphous with the native ones, whereas the crystals of the mutant may have somewhat different p acking. After resolving the SIR phase ambiguity by solvent flattening, the electron density shows a packing that is consistent with the nonc rystallographic symmetry found by Patterson searches as well as with t he motif observed in electron micrographs of thin sections of the crys tals. These studies established that phase information can be obtained from heavy metal clusters, even when the crystals under investigation are unstable and weakly diffracting. These results encouraged further effort at the construction of specifically derivatized crystals from other ribosomal particles that diffract to higher resolution. (C) 1995 John Wiley & Sons, Inc.