New Nucleating Strategy for Crystallography?

Well I was reading BK's excellent blog Life of a Lab Rat (an opinion piece from the Guardian "Only biology is safe and, as everybody knows, biology is science for girls." WTF?)

When I came upon a link to this great entry on x-ray crystallography (here is some background on what the hell x-ray crystallography is). My current lab is a remarkable mix of various disciplines in the life sciences ... we have biochemists, cell biologists and x-ray crystallographers. We non-crystallographers have had many discussions about that last group. They are a curious breed of biologists ... well really chemists. Some of it is puzzlement, some of it is wonder ("how could it be that none of them can subclone?")... but most is pity. You see unlike us biochemists and cell biologists, x-ray crystallographers can't make small incremental advances. Their projects are truly sigmoidal, the rate limiting step, as all you lab rats know, is getting the damn protein to crystallize. All this pain has led to a bizarre hierarchy within the field. You see not every x-ray crystal structure is equivalent. Certain structures are judged "more worthy" than others. So if the protein is involved in some disease or some hot topic (like RNAi) then your structure can (rightfully so) be published in a top-tier journal. But if the protein is hard to deal with then it also (in my opinion wrongfully so) can be published in a good journal. Call this aspect of a structure, the macho factor.

But lets not get sidetracked ...

My pity is so deep that I often brainstorm with one structural biologist in the lab (he does both crystallography and NMR) on how to solve all of x-ray crystallography's problems by forcing proteins to rearrange themselves in regular arrays. But we haven't come up with anything good strategy yet. How painful is it? Enter BK:

One of the things I was taught when I learned crystallography was to be very, very clean. No dust, human hair, bits of glass or other muck were allowed into the experiment. The most frustrating thing about crystallography is that all proteins are different, and will crystallize (if they crystallize) under different conditions, and there doesn't seem to be any pattern to this at all. The lab's insistence on cleanliness was an attempt to factor out one of the variables in the process. But I soon discovered that this may have been counter-productive. As with a lot of things in research, people disagreed with each other and there was a lot of intuition and opinion without a great deal of solid evidence. I realize that this might come as a surprise to some of you, to those who, perhaps, believe that 'scientists deal with facts'. The truth is that at the frontiers of science we don't know what's going on and we're trying to find out - that's why it's called 'research'. If you want facts, look in a text book (and they all contain mistakes, too).

So as I went on and got more experienced, I began to welcome small amounts of crud in my crystallization experiments. In fact, one recalcitrant protein only ever crystallized once, along what looked like an insect leg. I was never able to repeat that experiment; although I did have gothic fantasies about breeding every different sort of insect I could find and using various bodily insect parts as nucleants. A little too Shelley, perhaps.

BK then goes on to point on a new theoretical technique to nucleate protein crystals. If you can kick start the crystalization reaction (i.e. "nucleate") then you have a greater chance of getting the damn thing going. Again BK:

Strangely enough, the theory they've developed is for a disordered porous medium. The idea of a crystal is that it's highly ordered, for reasons that I won't bore you with here. I'm guessing that disordered means that you have a near-infinite number of different shapes in the medium, which means you've got more chance of finding the right shape you need to get that critical first nucleus. I do need to read the paper, don't I?

If this really works, maybe then I can stop feeling sorry for our crystallographers (although who can feel sorry for crystallographers when they get the cover of Nature?)

Ref:
Naomi E. Chayen, Emmanuel Saridakis, and Richard P. Sear
Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium.
PNAS (06) 103:597-601

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I think beer works fairly well...oh, wait, that's a nucleating strategy for crystallographers

I once tried adding champagne to crystallization trials. Just think of being able to charge bubbly as a legitimate lab expense. No, it didn't work.

By somnilista, FCD (not verified) on 29 Aug 2006 #permalink

I do know a lab that used cat whiskers as a nucleating agent. The trick was though it had to be the whiskers from one cat a lab member had and not another cat. Just crazy.

The motto in the lab where I learned crystallography was "Try everything". Not so useful early on in grad school but, unfortunately, true in crystallography. My first crystals grew in my first screen during a rotation. Other proteins have resisted thousands of attempts, doesn't mean it won't work eventually, I just didn't try everything.

I would take exception to your argument that difficult means top journal. I don't believe this is the case anymore and I'd be interested in some examples. The truth is that simply having a structure is much harder to publish, one really needs to have a biological story to apply to it. Fortunately(?), these days the really interesting problems are the tough ones so the big papers all have a healthy dose of biology and chemistry.

FadL? (and this strange obsession to crystallize membrane proteins ...)

Yeah, I thought about that one too. I think that was more along the lines of your earlier discussions about name recognition counting more than good science. I can tell you that no crystallographer thinks of outer membrane proteins as difficult to crystallize. They can easily be purified from inclusion bodies and can be refolded, a sign that they are easy to work with. FadL had a new fold (a unique number of beta-strands) and was the structure of a new type of transporter, still...

The obsession to work on membrane proteins is related to the fact that they represent the new fronteir in structure. Despite recent advances we really know very little about these very important proteins.

Well, thanks to Alex for the kind words. I've little to add (except damn, in a strange sort of way I miss setting up crystal screens), but kstrna's comment intrigues me.

Are you sure the cat's whiskers were being used to nucleate? Or were they using them to seed ('streak') from an existing crystal into a fresh drop? This is a neat technique for trying different growth conditions once you've got past the nucleation step and want to grow large(r) crystals. If you are right, and they were using whiskers for the first nucleating step, then hmm . . .

If lots of cats in Sydney start disappearing, you'll know why.

PS Alex, I think Emma was being slightly tongue-in-cheek when she made the comment about biology being for girls.

By Black Knight (not verified) on 29 Aug 2006 #permalink

It is not about protein crystallography, but cat whiskers reminded me of the following story.

The Physics of Snowflakes

In the 1930's, the Japanese physicist Ukichiro Nakaya set out to study ice formation in the laboratory, attempting to grow crystals suspended on a thread. Individual specimens proved elusive however, for the threads became encrusted with frost. Nakaya tried strings of cotton and silk, he experimented with wires and spider's web. Eventually he found the solution in a strand of rabbit's fur, where natural oils discouraged the nucleation of frost and allowed the development of isolated flakes. The result of this research was his famous snowflake morphology diagram.

nice...

I "dabble" in crystallography (when I want to take time out of my more "incremental biochemical/cell biological advances"), and keep trying to crystallize the proteins I work on.

I have amongst the most non-reproducable crystals around. Sometimes, I get dozens of crystals, but the same protein, purified the same way, crystallized in the same solutions, often gives me nothing.

I'm happy with my biochemical life :-)

I do know a lab that used cat whiskers as a nucleating agent.

I know that lab too (the Steitz Lab) but as someone else mentioned I think that they use the whiskers to seed crystals.