It's Cold In Here

Or, One More Reason to Take Protein-Ligand Crystal Structures with A Grain of Salt


This is going to be a really short post. Sorry, folks.

Ashutosh pointed out the other week to be wary of ligands in the Protein Data Bank (PDB) – see here. This is of course good advice, and everyone should read that post on the off chance they haven't already done so. Done? Good!


One other thing that I think is underappreciated by those who aren't protein crystallographers/structural biologists/biophysical BAMFs is that, for the most part, modern (synchrotron) protein crystallography is done under cryogenic conditions. Now, this typically is a bit warmer than the boiling point of liquid nitrogen, so you've got a protein crystal wallowing around somewhere above 90 K but not too far above 150 K. I'm not going to say that this is an entirely bad thing – it minimizes radiation damage, allowing for the higher-flux radiation sources to do their job. This of course can lead to higher-resolution structures and most of us generally appreciate that. However, here's the question – does it accurately represent the protein-ligand complex under physiological conditions?

A really interesting analysis of this question was addressed a few years back in 2004 by Bertil Halle from Lund University. I would recommend reading the paper, but if you're interested in the abbreviated conclusions, to wit:

1.)Flash-cooling of protein crystals – and subsequent cryocrystallography – is capable of retaining the general backbone fold and positioning of the protein, but

2.)The quenching of the solvent/ligand/ion degrees of freedom are not necessarily an accurate representation of the complex under physiological conditions and are probably more indicative of the system at the glass transition.

So the next time you look at a crystal structure and wonder, “How in Hades did that ligand end up there?” think about this. For a macroscopic analogy, consider the following structure:



Now, if it were 50 degrees colder, while the overall positioning would be similar, there's no reason to expect that every finger, strand of hair, and toe would be in the same position.

Expect more thoughtful posting one of these days, but don't hold your breath....

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Assorted Awesomeness.

So, I was going to do a post on general bad-assery and things I've found fascinating in magnetic resonance this time around, but it's taking a bit longer due to real-life laboratory fun. I will probably rework it as a really long bibliographic post, and perhaps have some discussion about it here and there. Onto the real-life laboratory fun, with apologies to Gossip Girl....


So, I've been aware of surface plasmon resonance (SPR) as a method for investigating kinetics and thermodynamics for a while now, and generally thought it was an interesting application. You can hardly do any reading in the biochemical literature and not eventually stumble across a paper that uses it. However, it was one of those things which I had never actually done until starting my current position.

Well, in order to further preserve some ostensible anonymity, I will be avoiding explicit details of my current work. The particular interaction I've been thinking about a lot as of late has been between this relatively small (~ 40 kDa) protein which I will call Blair, and this larger oligomeric protein that I will call Serena. Now, Serena prefers to relax in an appropriately tailored buffer, as is not surprising. So while I lovingly prepared Serena in such a buffer, I figured that for the purposes of the SPR experiment, I could just squeeze her into a relatively analogous buffer (minimal change in pH, no dramatic differences in overall ionic strength) that was a standard for doing SPR. I didn't think she'd mind, after all, she can retain her hotness under many conditions, or so I was led to believe.

Well, suffice it to say, after three rounds of SPR, I was mistaken. Now, I've learned how to finally run the instrument like a pro, I know that I need to go above and beyond the minimum for filtering my solutions and centrifuging out the dust in my samples, and can rewrite the scripting code half-drunk (not that I have, just that I can, it's really easy). There were other likely culprits (some potential issues with my controls, some concerns about coating densities) which I eliminated from the list of suspects the second and third times I ran the experiments, so I was making progress there. I just need to tweak the buffer conditions so Serena doesn't get all broken-down and sad on me. Because a hot blonde with great legs deserves better, metaphorically speaking.

Because, after all, how can you not want to see this?



Metaphorically speaking, of course.

One minor MR-related thing that will make my next post all about the spectroscopy – I find the idea of using nuclei other than ¹H in MRI to be endlessly fascinating. I'm very intrigued by the work people have been doing with, in particular, ²³Na – everything from cardiovascular physiology to neurological imaging to muscloskeletal studies. Not to say that I don't spread my love around for all NMR-active nuclei – certainly I'm not the only one to check out this absolutely fantastic website by Pascal Man periodically just to see what's new in the world of quadrupolar NMR – but ²³Na MRI seems to be a bit ahead of the pack, just from my anecdotal observations, in terms of development and rate of progress at the moment. If any MRI pros are out there, I'd love to hear from you, as I am positive I am missing out on some really neat things.

Well, that will be my obliged post for the week. I am going to strive for two posts next week. But don't hold your breath.....

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