September 28, 2010 00:57
Over the years there have been a number of Nobel Prizes awarded to scientists that have used their protein crystallization skills to provide unprecedented insight, usually at atomic resolution, into important biological processes. In appreciation of their contribution I had put these 12 crystallization heroes into the Protein Crystallizers Hall of Fame with the crystallization and structure determination of these proteins: Ribosome (2009), Water & Ion Channels (2003), RNA Polymerase (2006), Photosynthetic Reaction Centre (1988), Ion transporters (1997).
Which are the possible Protein Crystallization-related Nobel Prizes this year? What do you think - which of these target areas would you consider for a 2010 Nobel Prize (Chemistry or Medicine)?
Or here if you prefer to vote via your LinkedIn account:
There are plenty of reasons to select these protein structures since they have provided useful insight into biological function and should therefore be worthy a 2010 Nobel Prize:
Heat Shock proteins such as HSP90
Signal Transduction Molecules such as G-Protein Coupled Receptors
Molecular Pumps such as P-glycoprotein, Multidrug Resistance Transporters , or sodium/potassium pumps
Ion channel receptors such as the Acetylcholine Receptor and Mechanosensitive Channels Ubiquitin tagging system:
Viruses: Dengue , Tobacco Mosaic Virus , Parvovirus
Let's keep in mind though, that the Nobel Committee may take a break from structural biology in 2010 and focus on these areas that come to mind:
□ Discoveries of pluripotent stem cells / dentritic cells
□ Technologies: human genomics, sequencing, DNA microarrays
□ Pathways and Drugs: Leptin, DNA metallo intercalators
Now, with the 2010 Nobel Prize announcements coming up next week I'm keeping my fingers crossed for yet another award for this fine scientific craft. The announcements are expected to be made on
Monday 4 October, 11:30 a.m. Central European Time for Physiology or Medicine, and on Wednesday 6 October, 11:45 a.m. Central European Time for Chemistry
Regardless of the outcome next week, it's definitely an occasion that's worth getting a bottle of champagne out of fridge!
September 22, 2010 02:19
I've said it more than once: "5 min surfing the web can save you a week in the lab". Here's my most recent online favourite:
BRENDA, the Comprehensive Enzyme Information System.
BRENDA stands for Braunschweig Enzyme Database and its utility as a starting point for expression, purification and crystallization may be enormous if you're working on enzymes. The search function of this online database is simple to use, yet allows simple identification according to EC number, common/recommended names, systematic names and synonymous names.
Let's say you're interested in 'phosphatases'. Use this keyword to search under 'crystallization' and you'll be treated with a well-organized table that lists 16 different phosphatases with crystallization notes and references to the corresponding primary literature (see below). This works best for enzymes that have been crystallized already but you may be able to spot trends if your crystallization target belongs to a similar-behaving protein family.
The real strength of BRENDA though is additional information that is provided, such as stability information (pH, temperature, organic solvent, oxidation, storage) and functional parameters and binding partners (small molecule ligands, metals, substrates). This knowledge can be key to preparing the protein sample upstream of the crystallization process.
My take: if you're working on an enzyme and have not checked out BRENDA yet, go there today.
September 14, 2010 15:30
In case you have not heard about Emerald's newest protein crystallization tool, check out the MPCS - the Microcapillary Protein Crystallization System. This is an award winning, simple-to-use crystallization system that enables protein crystal growth with very little protein sample volume requirements (2.5 uL and you're in business). We have recently completed a side-by-side protein crystallization study and report our results in this paper:
Cory J. Gerdts, Glenn L. Stahl, Alberto Napuli, Bart Staker, Jan Abendroth, Thomas E. Edwards, Peter Myler, Wesley Van Voorhis, Peter Nollert and Lance J. StewartNanovolume optimization of protein crystal growth using the microcapillary protein crystallization systemJournal of Applied Crystallography (2010). 43
This is what the MPCS delivered in terms of successful crystallizations:
- 28 out of 29 (93%) proteins crystallized as compared to traditional vapor diffusion experiments
- 90 out of 120 (75%) protein/precipitant combinations lead to initial crystal hits from vapor diffusion experiments
- Many of the resulting crystals produced high-quality X-ray diffraction data, leading to six novel protein structures that were derived from crystals harvested from MPCS CrystalCards.
Protein crystallization study using the Plugmaker instrument and Crystal Cards.
The MPCS rocks,
September 10, 2010 19:39
There seems to be a live webcast from the ICCBM13, the 13th International Conference on the Crystallization of Biological Macromolecules at Trinity College in Dublin, Ireland. Stay tuned from 12th to the 16th September 2010 to learn what's new in protein crystallization science.
I'm not planning on staying up late over here on the west coast of the US. But the talks should be avalable for download at a later time, wouldn't you think so?
September 7, 2010 15:16
There's plenty of time to get all of your primary protein crystallization experiments prepared within a single day, including preparation for follow-up optimization experiments. Here's how you may want to schedule your protein crystallization day:
- First thing in the morning: get the protein out of the dialysis bag (or cassette) and transfer into the concentrator device of your choice. Save a small sample to determine the concentration:
- While the sample is concentrating measure its light absorption at 280nm, using the dialysis buffer as a blank. No need to run a calibration curve if you just shoot for 20 OD, i.e. concentrate your protein until the absorption at 280 nm has reached a total of 20 OD units. See here for how to go about this and what's behind this rule-of thumb.
- Once concentrated to 20 OD, quickly filter your protein sample with a filter, for instance a 0.1 micron filter to clean up the sample.
Depending on when you start your day in the lab, you will have the protein sample ready to go before noon or early in the afternoon. Now you've only got an hour or so to prepare the entire crystallization trial:
4. Setting up a single tray of sitting drop/vapour diffusion crystallizations takes less than 20 min, four trays should take less than one hour. Definitely include a sparse matrix screen such as the two tried-and-proven 96-well Wizard screens (Wizard I&II and Wizard III&IV).
5. If you've got 300 uL of protein sample volume, first use ca. 160 uL to prepare 4 trays and snap freeze the remaining volume in four aliquots without adding any cryo protectant once you're done setting up the crystallization trial (check out the best-practice snap freezing procedure here). Those aliquots will be useful for optimization of any hits or for reproducing the crystals that you get in this first screening pass.
6. Take a quick look at the first tray right after you have set it up and check if there's quick precipitation in many of the wells. If more than 1/2 of all the wells are cloudy, you may want to reduce the protein concentration by 1/3 or 1/2 for the remainder of the crystallization trial.
7. Note everything down what you did, especially any screw-ups and unusual observations. These notes can help you in strategizing further crystallization attempts.
At this time it's time of a cup of tea.
Have a good crystallization day,