Tips for Using Micromounts™ for Macromolecular Crystallography
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MicroMounts are used in much the same way as nylon loop mounts, although there are some important differences. The nonmagnetic stainless steel pins are compatible with existing bases and mounting hardware. Our suggested procedures follow. If you find better or novel ways to use them, please tell us!
Select a mount with a sample aperture size slightly smaller than the crystal size. Unlike with loops, you don't want to trap the crystal inside a liquid meniscus spanning the aperture. You want to have it resting between the edges of the aperture, to minimize excess liquid and background X-ray scatter.
Cut the pin (if necessary) to a desired length using our pin cutters or other cutters designed for spring-temper steel. The center-to-center distance between black lines on the pin is 2 mm. Use the millimeter scale on the top of the box to help cut it to the desired length, lining up the end of the pin with the right end of the scale.
Insert the pin into a standard goniometer base using a small amount of, e.g., Dow-Corning #976V high vacuum grease or Duco Cement hold it in place. For easier handling, attach the base to a magnetic rod. The pin can also be inserted into a 0.7 mm mechanical pencil.
Slowly and carefully insert the gold-colored polyimide into the crystal-containing drop, trying to minimize fluid disturbances and motion in the drop. Slide the sample aperture under your crystal, and then carefully remove the crystal + mount from the drop, keeping the crystal centered over the sample aperture.
To retrieve crystals that have settled to the bottom of a crystallization plate, try pressing downward to bend the tip of the mount so that it slides flat along the bottom.
If there is excess liquid, slowly and carefully insert a size 15 paper wick into the large opening that connects to the sample hole. If you have lots of liquid and you insert the wick too quickly, the crystal will flow with the liquid to the wick. For viscous liquids you may need to "mop up" around the crystal with the wick.
You should find that it is no longer necessary to use Paratone or other heavy oils to replace surrounding liquid that otherwise would form hexagonal ice rings. Mounted correctly, you should have very little liquid around your crystal - far less than when using nylon loops. The flash cooling rate will be much faster than in a loop and the cryoprotectant concentrations required to prevent icing much smaller.
In general, it's bad to have a lot of any liquid - water or oil - around your crystal during flash cooling: when the liquid freezes, it will tend to crush your crystal. If you have just a thin layer of liquid around the crystal, it will shatter (like the shell of an egg) and so do little damage.
If your penetrating cryoprotectant isn't enough to keep the external liquid from crystallizing, try a quick swipe through a low-viscosity oil or a solution containing a non-penetrating cryoprotectant (e.g., a large MW PEG) and then removing as much of the excess liquid as you can.
Flash cool the crystal by your favorite method. We recommend plunge cooling in liquid nitrogen, in liquid nitrogen that has been vacuum-pumped to near its freezing temperature (to reduce boiling), or (better but more dangerous) in liquid propane.
Tips for Mounting Very Small Crystals
When working with very small crystals, dehydration is your worst enemy and time is of the essence. We recommend working in a humidified environment or a cold room, or else using a very slow flow of cooled and/or humidified air directed at your work area. If you've ever compared how quickly puddles evaporate in the summer and in the winter (on sunny days on equal side of the solstices), you know that evaporation rates vary strongly with temperature. The saturated vapor pressure of water at 4°C is 1/4 that at 25°C.
Some users have complained that the mounts seem to push small crystals away, as if they were too hydrophobic. That's not what's going on. What you see is a simple demonstration of laminar flow and Stoke's Law. When you move the mount through a crystal-containing drop, the liquid flows laminarly around it. If the crystal's density matched that of the liquid, it, too, would just flow around the mount, and it would nearly impossible to snag it.
If the crystal's density is larger than that of the liquid, the crystal will sediment under the influence gravity towards the mount. The crystal's sedimentation speed is determined by the balance between the gravitational force pulling down and the viscous drag force given by Stoke's law that opposes motion.
It's an easy matter to show that this sedimentation speed varies as the square of the crystal diameter. Thus, the time for the crystal to sediment onto the mount increases rapidly as the crystal gets smaller. You then need to move the mount very slowly to allow enough time for the crystal to sediment through the liquid around the mount and onto its surface. For the smallest crystals, you should probably hold the mount with some sort of micromanipulator unless you have very steady hands.