The suitability of a monofunctional reagent of an undecagold cluster for phasing data collected from the large ribosomal subunits from Bacillus stearothermophilus.

Abstract

An electron density map of the large ribosomal subunit from Bacillus stearothermophilus was obtained at 26 A resolution by single isomorphous replacement (SIR) from a derivative formed by specific quantitative labeling with a dense undecagold cluster. For derivatization, a monofunctional reagent of this cluster was bound to a sulfhydryl group of a purified ribosomal protein, which was in turn reconstituted with core particles of a mutant lacking this protein. The native, mutated, and derivatized 50S ribosomal subunits crystallize under the same conditions in the same space group. Under favorable conditions, crystals of the derivatized subunit proved to be isomorphous with the native ones, whereas the crystals of the mutant may have somewhat different packing. After resolving the SIR phase ambiguity by solvent flattening, the electron density shows a packing that is consistent with the noncrystallographic symmetry found by Patterson searches as well as with the motif observed in electron micrographs of thin sections of the crystals. 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.