December 30, 2009 02:24
Acta Cryst F is the place to go to check out images of new protein crystals. I've just recently noticed that every single crystallization report is graced with a crystal. Way to go Acta Cryst F!
Cover illustration of the Jan 2010 issue of Acta Cryst F.
While browsing the new Jan 2010 issue I saw in their Notes for Authors 2010 That Acta Crystallographica Section F requires that Validation Reports need to be submitted for publication:
11.5. Validation reports
Authors of structural papers are required to provide a validation report on submission. Authors are encouraged to presubmit their data to the PDB and obtain a validation report for their structure.
Howard Einspahr and Manfred Weiss then go on to make a point in their editorial that this requirement will be fully enforced in 2010. I suppose that this is a direct consequence of the Krishna Murthy case and the associated recent crystal structure retractions.
Looking at the pretty pictures of protein crystals I realize that the only thing that can be possibly wrong with crystal pictures is that there's a mix-up (intentionally or not). There may be less damage with such mix-ups though, since the value of a crystal images is limited compared to all the other data (or isn't it?).
One thing is clear: each image of a new protein crystal is a gem that's worth showing off.
Happy new 2010,
December 24, 2009 15:00
The end of 2009 is near and it's time to clean up to make room for new and exciting projects in 2010. I'm just returning from the lab with a stack of protein crystallization trays to toss. Some of the crystallization experiments were prepared more than a year ago! There were two categories of crystallization trays that I dealt with:
1. Trays set up with protein that have never yielded any crystals at all
Should I keep them and hope that via slow desiccation or proteolytic cleavage protein crystals will eventually form? Nah! Everything that's older than 6 months must go. Gone they are. - I'm having second thoughts though, now that I'm writing this. I could open the crystallization chambers for a while and let the drops dry out just a little and then close them again. After all, protein crystallization by dehydration does work sometimes. Or move the trials to a different temperature? Or add chemotrypsin to the drops and create target fragments that crystallize.
Crystallizers' remorse setting in big time....
2. Trays set up with protein that have yielded crystals and structures
I could throw them out altogether. Crystals diffracted, structure is done. But, why not keep a few trays with crystals? You never know when a project 'comes back' - with the need to co-crystallize together with a small molecule ligand or protein partner. Even if the crystals don't diffract they may be useful and serve as seeds.
Pretty (Ammonium sulfate) crystals. These are the easy protein crystallization setups to give up on and throw out.
Phew! - that was easy, actually.
2010 here I come!
December 23, 2009 02:47
In many high-throughput protein crystallization laboratories protein crystallization is dealt with as a standard process that usually goes like this:
1. Search for protein crystallization condition using a standard array of crystallization reagent matrices (BTW - many labs use Wizard I, II, III and IV for such a first pass ;). These experiments are of the trial-and-error type, sparse matrix screening that is akin to shooting in the dark. Protein crystallizaiton trials are set up with volumes as low as the liquid dispensing tools at hand allow and plates for sitting (and, less frequently) hanging drops in fairly standardized way (i.e. combining 300 uL protein solution + 300 uL precipitant). Plates are then stored in the dark at one or two temperatures and inspected after a day, several days and weeks. Once crystals or crystal-like objects are identified, the next step concerns
2. Optimizing the protein crystallization condition to grow better X-ray diffracting protein crystals. At this stage the repertoire of protein crystallization optimization procedures explodes, and one needs to consider available resources, experience and the protein at hand to apply them best. Popular crystallization optimization procedures include:
• Systematic grid screening
• Temperature variation
• Additive screening
The good news about these optimization procedures is that they are systematic and the results of the crystallization optimization experiments are often informative because they point to trends. For instance, you can identify a temperature that produces larger protein crystals. However, these optimization experiments need to be carried out in a fairly disciplined way, as one dimensional variations of a single parameter, keeping all other parameters constant. The alternative would be screening an astronomical number of crystallization conditions:
There are only a few points in the multi-dimensional protein crystallization space that one visits in typical protein crystallization experiments. Looks daunting, doesn't it?
And here's the problem with this scheme (not that I have a solution to it, but I'd like to point it out nevertheless):
By screening just one dimension at a time, large spaces of the multidimensional crystallization phase space remain unseen. This I call the Curse of Dimensionality in protein crystallization. I'm wondering if there are there any practical ways to getting around it. Any ideas?
There's no need to get too tripped up with this, though. The good news is that most proteins can be made to crystallize. In my mind this means that we have quite some slack in defining protein crystallization conditions.
December 18, 2009 03:47
This is somewhat of a response to an issue brought up by Sean over at p212121. He left a remark, saying that there is only little to find in the technical literature discussing the practical side 'if this doesn't work, try this'. Having thought about it for a while, I think that the literature may not be the best place to look for such information.
Nevertheless, there's some 'tips-and-tricks'-type guidance on the topic of protein crystallization and crystallography as a whole. Protein crystallization in particular is a moving target since technologies develop and bottlenecks have shifted over time. Also, I've noticed that there's been a gradual shift in some publications picking up more of the detailed nitty-gritty. My all-time favorite of such publications is the "Methods in Enzymology" series that are a treasure troves when it comes to detailed methods descriptions. Thanks to Google you may find the relevant sections online for those that don't have online or library access to these books.
I also found the "Current Topics in...." series useful and the recently started journal "Nature Methods". See for instance Naomi's review on protein crystallization (rocks!).
I have been glad to see several - freely available - online supplemental sections in both Science and Nature magazines, giving detailed experimental background that was inaccessible or hidden in more obscure journals years ago.
Really, the best source of info for your particular protein crystallization case may come out of a discussion with a collegue and be more appropriately discussed at a scientific conference, over a beer (or both).
A month in the lab can save you a quick trip to the library.
December 16, 2009 02:19
Taking a wrong turn while hiking the great outdoors can be the start of a big adventure, but in the protein crystallization lab it usually means a waste of time, especially when dealing with optimizing protein crystallization hits. The critical point is a typical go/no-go decision where the question you need to answer is this: is it worthwhile to follow up on this particular crystallization experiment? The answer is "yes" if there are no other, potentially better crystallization hits and if you're sure that the objects you're looking at are protein crystals, though not yet useful for X-ray diffraction experiments. The result of a positive decision will be committing to an often tedious protein crystal optimization project, possibly weeks of re-purification and preparing numerous small variations of that protein crystallization setup.
At this point there's one very simple observation that you can make to avoid taking a wrong turn: check the reservoir solution for crystals. Any crystals there resemble those in the drop?
Since many crystallization cocktails are formulated with high salt concentrations, just a little dehydration can cause salt crystals to form.
So, if there are crystals in the reservoir you're very likely dealing with salt crystals in your drop and not the sought-after protein crystals. Don't take this turn and keep on searching for a better protein crystallization hit.
Good reasons to curb your enthusiasm: crystals in the reservoir solution of (A) hanging drop protein crystallization experiments or in (B) sitting drop protein crystallization setups are a harbinger of bad news: salt crystals.