Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting

Abstract

Unit-cell reproducibility is improved by maintaining the humidity of crystals during cryomounting.

Efficient cryoprotection of macromolecular crystals using vapor diffusion of volatile alcohols

“…The approach was effective for eliminating ice from well-diffracting crystals as well as the complete cryoprotection of crystals for which the negative control destroyed the crystal lattice. Initially, in-vial equilibration times of tens of minutes were used, since we found previously that small unit cell changes occur on that time scale for vial mounts of thaumatin crystals (Farley and Juers, 2014). Subsequently, for most of the proteins we tested shorter equilibrations (except for trigonal trypsin for which we only had two crystals) finding 10 seconds – 3 minutes produced high quality diffraction.…”

“…These enhancements have clear benefits, including improved reproducibility of cell parameters, the possibility of long in-vial incubations, more time for crystal handling, and more reliable removal of external solution (Farley and Juers, 2014). Simpler approaches were also tested with lysozyme, glucose isomerase, thaumatin and tetragonal thermolysin.…”

“…for insulin from 77.9 Å vs 77.6 Å for 45 second and 30 min incubations respectively). It should be noted that crystal packing changes associated with dehydration can occur slowly (Sanchez-Weatherby et al, 2009) (Farley and Juers, 2014). Therefore, in the event that short equilibrations are unsuccessful, we suggest that overnight incubations be considered, using an appropriately sealed vial/crystal cap.…”

“…Vial mounting proceeded as previously reported (Farley and Juers, 2014). Briefly, a cryovial (Hampton Research, Aliso Viejo, California, USA) was prepared by plugging the liquid nitrogen escape holes with clay and fitting an O-ring (amazon.com, nitrile rubber, 50A durometer hardness; 3/8” ID×1/16” thick) on the crystal cap (SPINE, Hampton Research).…”

“…Of the ~100,000 structures in the protein data bank, just 0.2% have methanol or ethanol present in the model, while 14% include either glycerol or ethylene glycol (Berman et al, 2000). A recently described vial mounting methods offers the possibility of turning the high vapor pressure into an advantage to deliver the volatile alcohol to a loop-mounted crystal (Farley and Juers, 2014). Here we show the approach is rapid and effective for several different protein crystals.…”