Amar and I pitched up last Thursday afternoon at the great gleaming doughnut in the Oxfordshire countryside with the latest batch of Amar’s crystals, packed carefully into a dry nitrogen dewar to keep the little jewels frozen at a chilly 80K. Because we were sharing the beamtime with other users from Imperial our slot didn’t start until about 8.30 in the evening. Amar had plenty of time to transfer the crystals — under liquid nitrogen — to the pucks that would eventually be loaded into the mounting robot. There was even time to stroll over to the cafeteria for a leisurely dinner.
But as soon as we got going with our experiments it was clear that something was not quite right with the samples. Each crystal was mounted in a tiny nylon loop stuck on the end of a thin metal pin. Plucked from liquid nitrogen by the robotic arm, they were kept frozen on the X-ray camera by a steady stream of nitrogen gas at about 100K. But rather than glinting in the fluorescent lights, the crystals looked opaque and rather ragged in outline.
That could mean only one thing: ice.
The diagnosis was confirmed as soon as the crystal was exposed to just half a second of the intense X-ray beam:
The concentric circular rings on the diffraction pattern, partially and annoyingly obscuring the lattice of spots that we needed to measure, were due to the presence of randomly oriented ice crystals, either within or on the surface of our protein crystal. Before freezing, protein crystals are usually soaked in a solution containing a cryo-protectant — or anti-freeze — such as glycerol. The cryo-protectant prevents the water within the solvent channels of the protein crystal from turning into crystalline ice. Instead it should form a glassy solid that only scatters X-rays diffusely and doesn’t seriously interfere with the diffraction pattern.
When you see ice rings it sometimes helps to “re-anneal” the crystal. You do this by blocking the cooling nitrogen stream for a few seconds, usually with a handy credit card, to allow the liquid surrounding the crystal to thaw and then quickly re-freeze it again.
Well we tried that. It didn’t work.
And then Jeremy, a colleague from Imperial, popped into the station and mentioned that Juan, the Diamond beamline scientist, had suggested ‘washing’ crystals with liquid nitrogen to get rid of ice.
By this time we’d been through several samples and were certainly open to suggestions. So with a long 25 mL plastic pipette dipped briefly into liquid nitrogen we quickly drizzled a few drops of the slick colourless fluid over the crystal mounted on the camera.
The effect was immediate. Already on the video screen the appearance of the crystal changed considerably. Gone were the ragged edges and out of the obscurity emerged a gleam of shining light.
Better still, when the crystal was exposed to X-rays the rings were gone. Completely.
And we could see that the crystals diffracted to about 1.5 Å. I’ve mentioned before what a rich treasure-tove of structural detail such data can reveal. Thanks to that timely tip-off from Jeremy and Juan, we were able to get stuck into several hours of solid, rewarding work. We were cooking, if you’ll pardon the pun.
When I got back to Imperial there was the usual round of inquiries as to how the trip had gone. It’s always good to be able to report that you got some data. But this time I was more interested in recounting the impressive effect of the liquid nitrogen wash – to spread the good news, so to speak. And I found plenty of eager ears.
And that’s what I’m doing here I guess. The title of this blog is Reciprocal Space, after all. Every so often I feel duty-bound to make some mention of X-ray crystallography and the joy it can bring.