Protein crystallization

It's time for an update on Foldit protein design! If you recall, our last update showed that several Foldit player-designed proteins appear folded and stable in solution. However, we'd like to have crystal structures of these proteins to show that they are indeed folding into their intended folds. The first step in getting a crystal structure is getting a protein crystal. Here we take a closer look at the protein crystallization process.

Above is a 96-well crystallization tray. We use a robot to rapidly set up crystallization experiments with 96 different conditions per tray. For this protein we set up four trays, to test a total of 384 crystallization conditions.

Each “well” in the 96-well tray is actually divided into four distinct regions. In the upper right, a square reservoir holds the mother liquor. The mother liquor is typically an aqueous buffer with some salt and a high concentration of precipitant. The reservoir is accompanied by three circular drop wells, each of which contains of drop of our protein sample mixed with the mother liquor. In this tray, the three drop wells are used to test different drop ratios, with protein and mother liquor combined in a ratio of 1:1, 2:1, or 1:2.

Each of the 96 wells is sealed off from the air and from neighboring wells. However, within a well, the three drops share an atmosphere with the reservoir, so that the drops can equilibrate with the reservoir by vapor diffusion. Over time, water evaporates from the drops and condenses in the reservoir. As the drop volume decreases, the protein concentration in the drop gradually increases. Eventually, the protein concentration reaches a critical point and the protein crystallizes.

In the drop above, we see several plate-like crystals radiating outward from a single origin. Most likely a small dust particle at the center served to “seed” the growth of all these crystals.

The crystals are not actually colored, per se, but exhibit birefringence—meaning that they refract light waves differently, depending on the orientation of the light waves with respect to the crystal lattice. When viewed through a microscope equipped with a light-polarizing filter, the birefringent crystals appear colored.

These crystals appear to be thin and plate-like, suggesting this particular crystal lattice extends readily in height and width, but less easily in depth. Sometimes, this is indicative of imperfections in the crystal packing, and can limit the quality of x-ray diffraction. To follow up, we’ll try to optimize the crystallization conditions by setting up a number of similar drops with slight alterations in the composition, in hopes that we get larger, more substantial crystals. However, there's a chance one of these crystals will diffract well enough to yield a crystal structure.

Once we have a nice, high-quality crystal that yields a good x-ray diffraction pattern, we can set about solving the crystal structure. A solved crystal structure will tell us definitively whether the protein folds up as the designer intended!

( Posted by  bkoep 51 371  |  Sat, 04/15/2017 - 00:09  |  8 comments )
Joined: 05/19/2009
Groups: Contenders
Thank you for the update !

Good to keep us updated (and motivated)

Joined: 03/05/2015
Groups: Gargleblasters
I agree... thanks... and please post more like this!

and please post more like this!

georg137's picture
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Protein crystallization

Many thanks. The overview of the process and techniques is a refreshing dose of reality after folding virtual molecules.

Joined: 06/06/2013
Groups: Gargleblasters

Keep us posted about those surfing hot dogs.... if there are folds that work and don't. Still undecided about ankle straps or seatbelts when I have time to do one of these :)

Joined: 09/24/2012
Groups: Go Science
waw !

It's ingenious.
It must be exciting to see a crystal appear.
Thanks for sharing this with us. It's quite motivating !

Susume's picture
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Is that a foldit protein?

Thanks for the explanation! Is the photo above an actual foldit protein, or some other protein that happened to be at the right stage for a photo? It's exciting to think we may be seeing a photo of a player design!

"Most likely a small dust particle at the center served to “seed” the growth of all these crystals." Are dust specks a planned part of the process? What happens if there isn't one?

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Protein crystallography used

Protein crystallography used to have the reputation of being a bit of a black art. Supposedly the most successful crystallographers had beards which were a repository for all kinds of dust particles which acted as seeds.

bkoep's picture
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Foldit protein crystal

The above protein crystal is indeed a Foldit player-designed protein!

Dust specks are not exactly "planned" here, but they can certainly play a role in the crystallization process. In general, nucleation is the most difficult step of the crystallization; once the beginnings of a crystal lattice are formed, the crystal will grow relatively quickly. Crystals can nucleate spontaneously, but it is also common to try to speed up the nucleation process. A crystallographer will often sacrifice an imperfect crystal, and break it apart to provide seeds for new crystals.

And I have also heard the legend of the bearded crystallographer, whose dandruff was especially effective for crystal nucleation...

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Developed by: UW Center for Game Science, UW Institute for Protein Design, Northeastern University, Vanderbilt University Meiler Lab, UC Davis
Supported by: DARPA, NSF, NIH, HHMI, Amazon, Microsoft, Adobe, Boehringer Ingelheim, RosettaCommons