Modified counterweights and beamstop for the Huber 512 and 511.1 four‐circle diffractometers

A novel method of heavy-atom equilibration with protein crystals is presented. This method utilizes a central drop of mother liquor containing the crystal surrounded by a locus of small drops containing the heavy-atom reagent. After slight differences in osmality damaged the crystals in a recent structure solution (Burley, David, Taylor & Lipscomb, 1990), we developed the following method for minimizing osmotic shock to crystals in heavy-atom search trials (Petsko, 1985). The method equilibrates crystals with heavy-atom solutions by vapor diffusion, using the American Crystallographic Association (ACA) crystal plates (Jones, Ward & Perrozo, 1986). A single ACA crystal plate holds up to 15 different equilibrations. Although one could use other plates and methods, the ACA crystal plate has optical clarity and lends itself to easy crystal manipulation, thus leading to a successful structure solution. The convenience of the sitting-drop mode was used for all of the manipulations. The reservoir had been previously filled with 500 Izl of mounting buffer. The crystal and its mother liquor were already in equilibrium with this buffer. A 20 ILl drop of mother liquor containing the crystal was placed in the center of a silane-coated coverslip. Around this, the heavy-atom-containing solution was placed in 2 ~1 droplets in a locus around the circumference of the crystal-containing drop (see Fig. 1). The heavy-atomsolution ionic strengths were not necessarily the same as that of the crystal-containing droplet at the beginning of * Current address: Department of Cell Biology, Stanford University Medical Center, Stanford, CA 94305, USA. the experiment. A top silane coverstilr then sealed the system and within 24 to 48 h the droplets were in osmotic equilibrium with each other as judged by two criteria: the droplet size (volume) had ceased to change and upon fusion of the menisci there were no observable density gradients. To merge the solutions, the top coverslip was removed and the solutions rapidly combined to avoid undesired evaporation from the solutions. The following sequence can be done in less than a minute. When combining the solutions, the droplets were slowly merged serially with the main drop while observing the process through a polarizing stereo microscope. If there was a density gradient upon fusion of the first menisci, the difference in density (osmality) was immediately obvious and further fusions were postponed until the equilibration was complete. During the addition of the reagent, the crystal was monitored closely through the microscope for changes in its polarization, which were indicative of immediate disorder. After fusing