by Peter Nollert
October 23, 2009 08:35
Your crystals are filled with water inside and they are wet on the outside. How do you cool a single crystal down to liquid nitrogen temperature without turning that water into hexagonal ice crystals? That's the prerequisite for macromolecular cryocrystallography: obtaining low-mosaicity X-ray diffraction data while avoiding the nasty 'ice rings' at 3.897, 3.669, 3.441, 2.671 and 2.249 Angstroms.
Here are my 5 Rules of Crystal Cryopreservation:
1. Small is beautiful. Larger crystals have lots of mass that takes time to cool. Smaller crystals cool faster. If you have enough X-ray flux, better go with the smaller crystals.
2. Soaking wet is bad. Try to wick away as much water from the crystal as possible. Avoid the big blob of water with crystal swimming around. Wicking away excess water by tapping on a dry surface has worked very well for me in several cases (e.g .tap on dry spot on glass cover slide next to the drop you're fishing the crystal out of). Dragging crystals through oil helped me a lot in a particularly stubborn case.
3. Cool fast. Minimize the time to go from drop to liquid nitrogen. It turns out that the last milliseconds before the crystal feels the liquid nitrogen are crucial. That's why Robert Thorne recommends puffing away that thick layer of insulating room temperature nitrogen gas and plunge the crystal quickly into the liquid nitrogen. He calls this hyperquenching.
4. Test, test, test. Test different cryo-reagents and procedures. In most cases you'll dip the crystal into a cryo-solution before cooling the crystal in liquid nitrogen. Testing different cryosolutions and methods will likely result in an optimal procedure for crypreservation. For inspiration check out Artem Evdokimov's nice simple and thorough recipe for cryoprotection of delicate crystals.
5. Laissez-faire. If you only want to check if the crystal you've got is a protein crystal (i.e. has many spots in patterns) and not something else: just go for it! Dip the crystal into the liquid nitrogen without any further ado and don't worry about the ice rings, just get the crystal into the beam and optimize cryo-conditions later.
Example for a hexagonal ice crystal. Not what you want to see when cryo-cooling protein crystals.
And here's my shameless plug: the smart way to pre-empt any of the above is to include the Emerald BioSystems' Cryo-screens into your primary crystallization screen repertoire. I've heard many crystallizers praise these screens. Any protein crystallization hit in Cryo I or Cryo II will cool in liq. nitrogen without creating any of the dreaded hexagonal ice diffraction patterns.
As always: wear your gloves and safety goggles when handling liquid nitrogen,
Peter