And then just drizzle some liquid nitrogen…

At the Diamond synchrotron last week, Jamie Oliver would have been proud of us.

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:

Diffraction with ice

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.

Diffraction without ice

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.

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28 Responses to And then just drizzle some liquid nitrogen…

  1. Sean Seaver says:

    Congrats! I have been around a number of liquid Nitrogen washings (unfortunately, since always feels like a last ditch effort) and have also had some success 🙂
    What are the pucks that you mention for transferring the crystals to the robot?

  2. Eva Amsen says:

    I was hoping this was going to be about making ice cream with liquid nitrogen. I was disappointed that it wasn’t, but then I read the crystal-washing story and still thought it was awesome!

  3. Wladimir Labeikovsky says:

    i love a story with a happy ending _. Does the N2 physically wash the ice crystals off?

  4. Richard P. Grant says:

    Ooh. I’ve successfully re-annealed crystals to improve resolution, but not heard of that trick.
    Mind you, I’ve never had water rings that bad. ( cough technique cough )

  5. Cameron Neylon says:

    I grant you that’s cool but how on earth does it work? You can’t presumably be actually washing the water out, it was already crystalline in the liquid N2 boil off so surely you’re not making the water amorphous by cooling? Hmmm

  6. Clare Dudman says:

    This made such exciting reading – I didn’t notice my husband come in just now. It is interesting how it works. I’d assumed that the water dissolved in the liquid nitrogen but I guess that wouldn’t work…

  7. Stephen Curry says:

    Morning all! Glad you enjoyed our little story…
    I was surprised at the success of the washing method. I presume in our case the problem was due to the accumulation of ice crystals on the outside of our protein crystal, not with the initial cryo-cooling. The crystals were stored in liquid nitrogen for up to a few days prior to our trip. They were then transferred to a ‘dry’ nitrogen dewar which kept them cool for transport. This dewar was then refilled with liquid nitrogen on arrival at the synchrotron. The crystals passed through a couple more baths of liquid nitrogen while being loaded into pucks and finally into the robot’s dewar.
    My guess is that at some stage the liquid nitrogen we had been using may have become contaminated with water ice. It is bloody cold after all so even water vapour from the air will condense within it. Perhaps such water ice crystals within the liquid nitrogen have a tendency to accumulate on the surface of our samples?
    This would explain why annealing (thaw & refreeze) didn’t work whereas washing did the trick. There was no ice within the protein crystal (which has large solvent channels between the protein molecules); if there had been the liquid nitrogen wash would never have dislodged it. (I’ve never had much luck with annealing, truth be told.)
    My bet (in agreement with Wladimir) is that the shear forces from the flow of nitrogen over the crystal during the wash were sufficient to remove the ice crystals stuck to the exterior of our protein crystal. This is fairly evident from the change in appearance of the crystal following the wash.
    I think in future we may need to be more careful with the dryness of our nitrogen – but any other suggestions will be most welcome.

  8. Stephen Curry says:

    @Sean – You asked about the pucks – did you mean what type? As I’m sure you know, the pucks are machined aluminium cylinders that (in this case) take 16 crystals mounted on pins. I confess I didn’t look so very closely at the robot on station I02 at Diamond but I think its’s a Rigaku instrument.
    I have a set of pucks that fit the robot at the ESRF but these are a different size to the ones used at Diamond so we had our crystal vials on canes and transferred them to the pucks at the beamline. They are a rather finicky design – you have to take the crystal pin out of the vial to insert it in the puck!

  9. Henry Gee says:

    What a great post, Stephen. Notwithstanding and inasmuch as which I knoweth not protein Xtallography from an hole in the ground, what got me was the sterling humanity of it, a tale well told, with excitement, disaster averted by the ingenuity of the protagonists, and a happy ending. I’m going to go now. Need Kleenex.

  10. Richard P. Grant says:

    Talking of Diamond, pretty!
    ‘Intense beams of electrons’, though? Not quite. I can see why they said that, but I’m guessing you can’t say X-Rays in the mainstream meeja these days.

  11. Stephen Curry says:

    Thanks Henry! Appreciate it.
    @Richard – yes it is a tad ambiguous. The electrons are accelerated by the synchrotron in order to produce the X-ray beams that do the actual probing, so it’s not inaccurate. However, there’s no ban on ‘X-ray’ per se since the term is used elsewhere in the article.

  12. Richard P. Grant says:

    Well… I don’t think the writer understands. Which means the average reader has no hope. And we should all start saying that synchrotron radiation is produced by electrons making handbrake turns at the speed of light.

  13. Richard Wintle says:

    What an excellent story, Stephen. I would wax eloquently about it, but Henry’s already done so to excellent effect.
    Crystallographers everywhere are filing that trick away, now, as a result of your post. I can sense it.

  14. Stephen Curry says:

    Thanks Richard, though I suspect the technique may already be reasonably well known. Sean Seaver (above) had certainly seen it. We’re not really pioneering but it’s good to spread the message!
    I wonder where it originated?

  15. Sean Seaver says:

    Ah, I was able to track down some info about pucks, neat. I have never had the chance to use a robotics system at a beam line.

  16. Stephen Curry says:

    I have never had the chance to use a robotics system at a beam line.
    Once you do, you’ll never look back. It’s great to be able to load up the robot with pucks — I think the one at Diamond takes 5 or 6, so that’s up to 96 crystals — you can withdraw from the hutch and run everything by computer.
    All we need now is a remote controlled liquid nitrogen squirter!
    I may live to regret this but I am looking forward to the day when just the crystals go to the synchrotron and we can run the experiment from home (or on the bus with my iPhone…)!

  17. Jennifer Rohn says:

    What a wonderful story, Stephen – my favorite kind of blog post. Incidentally, what’s that weird blobby thing on the left?

  18. Richard P. Grant says:

    bq. I may live to regret this but I am looking forward to the day when just the crystals go to the synchrotron and we can run the experiment from home (or on the bus with my iPhone…)!
    Um. It’s called Grenoble, Stephen.
    Jenny, that’s the ‘hat’ that the pin is attached to the goniometer via. See pic at

  19. Stephen Curry says:

    Thanks Jenny – I’ll bear that comment in mind for the future…
    Richard isn’t quite right about the blob on the left. The hat is further to the left and out of shot. The blob is actually the end of the thin metal pin (only about 0.2-3 mm in diameter) that the nylon loop holding the crystal is attached to.
    And Richard, yes I’ve heard that the ESRF in Grenoble offers a data collection service to industrial users but I guess it’s quite costly! I’ve never been offered it myself. We have beamtime there in December but will have to make the trek ourselves…

  20. Richard Wintle says:

    So, looking at the “thing that isn’t a hat” – are those the offending crystals I see all over the surface of it? The one in the lower (nice) diffraction image looks considerably smoother.

  21. Jennifer Rohn says:

    Exactly what I was thinking, but was worried that I was just seeing what I wanted to see…as usual.

  22. Richard P. Grant says:

    That’s odd Stephen, because we used to send crystals from the LMB to ESRF. And that weren’t industrial…
    (and yes, it’s the pin, not the hat. Doh).

  23. Stephen Curry says:

    @Richard W. & Jenny – yes the oval shape on the right (with the red target rectangle superposed on it) is the crystal in a blob of cryoprotectant within a nylon loop. In the top photo this looks rather raggedy because of the accumulation of ice crystals on it. It looks dark in that picture because the illumination is from behind, but in the light it has more of the appearance of a little snowball.
    @Richard G. – you’re right. Coincidentally, I was talking to an old friend from Oxford tonight who has used the ‘remote data collection facility’ at Grenoble but I’d not been aware of it before. Apparently you have to ask! Must look into it.

  24. Heather Etchevers says:

    What Eva said up above.
    You know you want to come to Grenoble in December – the skiing is great, if the snow has come early enough! I’ll get you a hot drink in Paris on the way, if you like.

  25. Juan Sanchez-Weatherby says:

    Dear Stephen,
    It’s Juan from Diamond. Thank you very much for letting people know about crystal washing I’m glad it is getting so many people talking and I’m glad it worked so well for you. The idea itself wasn’t mine – it has been circulating around Diamond for a while now.
    Just wanted to explain how and why crystal washing works, and to clarify some of the questions above. Firstly, washing works by removing ice that sometimes gets stuck to the surface of the crystals. Where the ice comes from is up for debate but it may form when samples are handled or stored, or during transit.
    The pucks we use at Diamond are UniPucks, see for details. They were chosen as they gave higher density and safety for transport. It is true that transferring samples from vials to these pucks requires some practice, but once mastered it is safe. In order to avoid problems we are working on developing a new tool to transfer samples in vials directly to the pucks with no handling. One of such tools is available now and several more will be available in early January. Check: for details.
    Funnily enough, today we were commissioning a LN2 pump that automatically washes crystals. The device is in place but it still needs some testing. Maybe next time you come along you won’t need a 25 ml pipette.
    Finally, another fun recipe for you: you can remove ice from LN2 and prevent it from getting to your crystals by filtering it with paper. The best is blue paper roll so that you can see the effect. 2-3 sheets work wonders! If you have been cryo-cooling samples and the LN2 starts getting icy just pass it through the paper and use it again. I always use this system when I refill the transport dewars.

  26. Stephen Curry says:

    Wow – many thanks for that Juan – a super informative comment (especially the tip about filtering the liquid nitrogen to remove ice)!
    I can appreciate the superior transport properties of the UniPucks since we’re just back from a trip to the ESRF and found a couple of loose crystal vials in the dewar on our arrival. No major harm done, thankfully.
    I’m glad to hear that the LN2 washer will soon be in place at Diamond – I presume this will be installed for all MX beamlines?

  27. Stephen Curry says:

    P.S. Heather – I’m afraid we didn’t pass through Paris on this trip. We’ll have coffee next time!

  28. Juan Sanchez-Weatherby says:

    At the moment only I02 will have a washer. Once we know how well it works we’ll roll it out to the others. Mid 2010 I expect.

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