Chae Un Kim
Cornell University, Ithaca NY
Abstract:
The flash cooling
of protein crystals is the best known method to effectively mitigate
radiation damage in macromolecular crystallography. To prevent physical
damage to crystals upon cooling, however, suitable cyroprotectants must
usually be found, a process that is time-consuming and, in certain cases
unsuccessful. Recently we have developed a novel method to cryocool protein
crystals without the need for penetrative cryoprotectants. In the new
method, each protein crystal is pressurized up to 200 MPa (2000 atm) in He
gas at 10 ˚C. The crystal is then cyrocooled under pressure and the pressure
was released while the crystal is kept cooled at 77 K. Results are presented
for several proteins that have been flash-cooled at ambient pressure and
pressure-cooled, in all case without penetrating cryoprotectants. For
example, the flash-cooled glucose isomerase crystal diffracted to only 5.0 Å
and mosaicity could not be estimated but the pressure-cooled one diffracted
to 1.05 Å with 0.39˚ mosaicity. For thaumatin, the flash-cooled crystal
diffracted to only 1.8 Å with 1.29˚ mosaicity but the pressure-cooled one
diffracted to 1.15 Å with 0.11˚ mosaicity. The protein structures show that
the structural perturbation by pressure is at the level of a few tenths of
an angstrom, which is comparable to the typical structural changes always
observed upon flash cooling at ambient pressure. A mechanism on the pressure
cooling is proposed involving the dynamics of water at high pressure and
high density amorphous (HDA) ice which is produced at high pressure and is
metastable at 100K. The potential applications of the high pressure cooling
method are discussed at the end of the talk.
2008 Run
Nov 19th - Dec 22nd
