by Peter Nollert
September 24, 2009 15:46
Sean Seaver over at P212121 recently blogged about the crystallization of his target protein in Emerald's new plug-based crystallization system, the MPCS (microcapillary protein crystallization system). Sean showed up with protein samples and crystallization formulations at our booth at the ACA in Toronto more than a month ago and Cory set up a crystallization trial in CrystalCards with the new PlugMaker instrument. This protein crystallization experiment consumed only a couple of microliters (each crystallization plug has a total volume of ca. 15 nl) while screening a multitude of slightly varying precipitation concentrations. I was happy to see images of crystals that had formed (images of these crystals are posted on Sean's blog). Pretty good for a crystallization trial that has traveled in a plane, took a long ride in his shirt pocket and made it to Alaska and back to the University of Toledo in Ohio.
Cory has left recommendations on how to proceed at P212121 - but since Sean has asked 2 questions that have come up previously, I'd like to address these here:
1. How can you tell which crystallization condition is in which drop?
This is a straight forward counting job: In a standard gradient run, a number of plugs are produced - let's say 200 individual crystallization experiments (=plugs), where the concentration of a precipitant is varied in a linear fashion (say from 0 to 100%). The start and end of the gradient are marked by longer plugs (with multiple gradients in a CrystalCard) or are defined by the length of the channel. In Sean's case we set up a shallow gradient of ca. 200 plugs (see a similar experiment in the figure below). Let's say you'd see a crystal in plug # 134, then the concentration of the precipitant in that particular plug is 68% (134/197) as compared to the maximum precipitation concentration used. If the maximum precipitation concentration was 2.5 M, the concentration in plug #134 is 1.70 M. Often crystals appear in neighboring plugs (this depends on the size of the 'crystallization slot'), which appears to be the case with Sean's experiment.
Figure. Identifying the accurate precipitation concentration in a particular plug within a CrystalCard (protein used is Thaumatin). A shallow gradient of 197 plugs with precipitation concentrations ranging from 0-2.5 M Sodium Potassium Tartrate was prepared. Plug #134 has a protein crystal, corresponding to 68% (134/197) = 0.6802, or 1.70 M of Sodium Potassium Tartrate.
2. How do I scale up?
There are two answers:
a) You don't need to scale up at all! Just take the crystal out of the card by removing the plastic laminate and fish with a loop as shown in this video. Alternatively, you can place the entire card into the X-ray beam and collect data that way. Check out these links about further info on in-situ X-ray diffraction of protein crystals within CrystalCards.
b) The plugs prepared by the MPCS are essentially small versions of batch-under oil crystallizations. A good way to mimic a plug is to combine a portion of the protein solution with precipitant at the concentration as figured out in #1 above and cover with oil. If you want to eliminate any effects caused by the plastic bottom of the crystallization tray and closer imitate the oil layer that completely surrounds the plugs in the CrystalCards, you may want to try container-less crystallization.
Let us know if there's anything else you'd like to know about this new low-volume protein crystallization system.
Peter