Learn Technotes Using MicromountsTM and MicroLoopsTM for Macromolecular Crystallography

TECHNOTES
Using Micromounts™ and MicroLoops™ for Macromolecular Crystallography

MicroMounts™ and MicroLoops™ are used in much the same way as nylon loop mounts, and are much easier to use than glass fibers and other mounting methods traditionally used in small-molecule crystallography. Their non-magnetic stainless steel rods are compatible with all existing goniometer bases (caps) and mounting hardware. Our suggested procedures follow.

Select a mount with a sample aperture size slightly smaller than the crystal size. Because competing nylon loops and mylar mounts produce so much more background diffraction than MiTeGen’s products, most crystallographers try to mount their crystal in the middle of the aperture, far from the nylon or mylar, and held in place by a liquid meniscus spanning the loop. This keeps the X-ray beam away from the strongly scattering nylon or mylar. But all that extra liquid boosts your background and can crush your crystal when it freezes. With MiTeGen’s products, you never have to worry about background from the X-ray beam hitting the mount.

Mount your crystal so that it rests on top of the aperture, so as to minimize excess liquid and associated background X-ray scatter. Choosing a slightly smaller aperture also makes it much easier to fish the crystal out of its solution. You don’t “loop” your crystal, you scoop it! (Of course, you can still mount your crystals the old way if you’re more comfortable with that, but our approach will usually give you better data.)

Insert the stainless steel rod of the MicroMount™ / MicroLoop™ into one of MiTeGen’s reusable goniometer bases, or else into a standard goniometer base using a small amount of, e.g., epoxy, glue or Dow-Corning #976V high vacuum grease to hold it in place. For easier handling, attach the base to a magnetic wand. The rod can also be inserted into a 0.7 mm mechanical pencil.

Slowly and carefully insert the gold-colored polyimide tip of the MicroMount™ / MicroLoop™ 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 if possible. It’s often helpful to brace the magnetic wand or pencil against your other hand or, e.g., a piece of 1/4″ thick foam to help steady the mount and allow smaller, slower motions.

To retrieve crystals that have settled to the bottom of a crystallization plate, try pressing downward to bend the tip of the mount against the bottom so that it slides flat along the bottom. You can often dislodge crystals with gentle pressure.

If using MicroMounts™, you can remove excess liquid by slowly and carefully inserting 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. Liquid can also be removed by touching a piece of absorbant paper to the back side of the aperture.

Mounted correctly, you should have very little liquid around your crystal. The flash cooling rate will be much faster and the cryoprotectant concentrations required to prevent icing much smaller. If there’s just a thin layer of liquid, it will tend to shatter (like the shell of an egg) on cooling rather than crush your sample.

To minimize ice formation on cooling and to keep your crystal from drying out during handling, first transfer it to a drop of our LV Cryo Oil™ and then mount it. If necessary, remove excess oil by wicking it away, or by flicking it away by gently tapping on the steel rod of the mount.

Flash cool the crystal by your favorite method. We recommend using the hyperquenching protocol of Warkentin et al. (J. Appl. Cryst. (2006) 39, 805-811) , a variant on standard plunge cooling in liquid nitrogen in which you you blow away the cold gas layer that forms above the liquid nitrogen just before you plunge in your sample. You can also use liquid nitrogen that has been vacuum-pumped to near its freezing temperature (to reduce boiling), or (more dangerous) liquid propane, but the hyperquenching method is safer, easier and generally gives the best results.
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