Protein Crystallization Hits

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
• Seeding

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.

Happy Holidays,
Peter

Your crystals are filled with water inside and they are wet on the outside. How do you cool a single crystal down to liquid nitrogen temperature without turning that water into hexagonal ice crystals? That's the prerequisite for macromolecular cryocrystallography: obtaining low-mosaicity X-ray diffraction data while avoiding the nasty 'ice rings' at 3.897, 3.669, 3.441, 2.671 and 2.249 Angstroms.
Here are my 5 Rules of Crystal Cryopreservation:

1. Small is beautiful. Larger crystals have lots of mass that takes time to cool. Smaller crystals cool faster. If you have enough X-ray flux, better go with the smaller crystals.

2. Soaking wet is bad. Try to wick away as much water from the crystal as possible. Avoid the big blob of water with crystal swimming around. Wicking away excess water by tapping on a dry surface has worked very well for me in several cases (e.g .tap on dry spot on glass cover slide next to the drop you're fishing the crystal out of). Dragging crystals through oil helped me a lot in a particularly stubborn case.

3. Cool fast. Minimize the time to go from drop to liquid nitrogen. It turns out that the last milliseconds before the crystal feels the liquid nitrogen are crucial. That's why Robert Thorne recommends puffing away that thick layer of insulating room temperature nitrogen gas and plunge the crystal quickly into the liquid nitrogen. He calls this hyperquenching.

4. Test, test, test. Test different cryo-reagents and procedures. In most cases you'll dip the crystal into a cryo-solution before cooling the crystal in liquid nitrogen. Testing different cryosolutions and methods will likely result in an optimal procedure for crypreservation. For inspiration check out Artem Evdokimov's nice simple and thorough recipe for cryoprotection of delicate crystals.

5. Laissez-faire. If you only want to check if the crystal you've got is a protein crystal (i.e. has many spots in patterns) and not something else: just go for it! Dip the crystal into the liquid nitrogen without any further ado and don't worry about the ice rings, just get the crystal into the beam and optimize cryo-conditions later. 

Example for a hexagonal ice crystal. Not what you want to see when cryo-cooling protein crystals.

And here's my shameless plug: the smart way to pre-empt any of the above is to include the Emerald BioSystems' Cryo-screens into your primary crystallization screen repertoire. I've heard many crystallizers praise these screens. Any protein crystallization hit in Cryo I or Cryo II will cool in liq. nitrogen without creating any of the dreaded hexagonal ice diffraction patterns.

As always: wear your gloves and safety goggles when handling liquid nitrogen,
Peter

In an ideal world, this would be a fairly simple way to grow protein crystals for X-ray structure determination purposes:

Start out with ca. 400 ul of filtered target protein solution, in 20 mM Hepes buffer neutral pH and maybe 50 mM NaCl , freshly purified to ca. 95% purity and concentrated to 10 mg/ml.

1. Snap freeze four x 25 ul aliquots of protein samples in thin-walled PCR tubes and store at -80C. These sample will be used in a week for protein crystallization optimization.

2. Document the quality of the protein sample via SDS-PAGE and determine its concentration (ca. 5 ul). Measuring UV absorption (OD280) and calculating the protein concentration is good enough.  You'll need ca. 5 ul for such an OD measurement (depending on cuvette path length and dilution). Check this online tool to get an estimate for the extinction coefficient based on sequence.

3. Now let's get out the multichannel pipettor and set up all of the remaining 200 ul of sample solution as 1ul + 1 ul sitting drop vapor diffusion crystallization experiments. A great first pass would look like this:
Plate 1: Wizard 1 & 2 (96 drops)
Plate 2: Wizard 3 & 4 (96 drops)
By the way, Emerald offers these reagents as the Wizard Suite. This is a 192 point, non-overlapping, sparse matrix that has a proven track record to yield crystals in first-pass protein crystallization exploration trials.

And while we're at it: go with the Compact Jr. plates. These plates can be sealed with clear tape and drops form nicely on the hyrdophobic polypropylene surfaces.

4. Store the crystallization trial at room temperature, minimizing temperature fluctuations and vibration. Observe right away, after 1 day, 3 days and 1 week.

5. The initially clear drops with now contain precipitate, some clear drops and a few with clustered microcrystals or needles in them. If the corresponding well-solution does not contain similar crystals, chances are that you've grown protein crystals! Since these crystals are likely to be too small to diffract them using your home-source X-ray generator and detector system, you'll need to optimize crystallization conditions and grow larger crystals.

6. There are many different ways to optimize crystallization conditons, and depending on prior knowledge you may want to carry out seeding experiments, include additives or change the treatment of the protein sample (i.e. filtration). Here's a simple optimization schema: create a grid screen around all conditions that gave you crystals. This rational crystallization optimization schema works great since it separates the effect of pH, salt, precipitant and protein concentration). You should use the online ScreenBuilder design tool to create an optimization screen. Your fellow researchers at Emerald BioSystems  are happy to prepare and send such a customized optimization screen to you.

7. Set up 96 - follow-up optimization 1 ul + 1 ul crystallization experiments with the saved protein material that you thaw in your fingers. If everything goes according to plan, crystals of different sizes will grow.

8. Harvest a crystal, cryoprotect, diffract and determine its structure ;)

Enjoy,

Peter

Need a simple way to test the effect of multiple temperatures on your protein crystallization trials? Take a thermometer and explore temperatures in your lab environment! There's plenty of evidence in the literature that temperature plays a role in successful protein crystallization. In some cases temperature is the crucial parameter to get right for growing well-diffracting crystals.
The BMCDB gives a good impression regarding temperatures that have worked well for protein crystallization in the past: 

Protein crystallization temperatures taken from BMCD 4.02. Note that the most favorite temperatures are those found in the lab and in a fridge.

I do think that the peaks at 4C and 20C are due to investigator bias and there's no scientific reasoning that these temperatures provide you with the highest crystallization success or best crystal quality. Since room temp. is so convenient, many labs underutilize this parameter as their primary way to optimize protein crystal growth.

Of course there are high-end incubators, but these are expensive and as a result, resourceful researchers have found cheap ways to access temperatures that are different from that on the lab bench.
If you want to explore more crystal growth temperatures for your protein crystallization experiments - get yourself a thermometer and go on a temperature hunt around you. How many different temperatures can you localize?
• Laboratory room temperature - that's easy, but there's potential to discover more than your standard 22C. You're in good shape if you've got an air conditioning system running, this provides you with a constant temperature. But even in air conditioned rooms you will often find spots that are warmer (i.e. on the top shelf, behind the fridge, on top of a warm instrument/monitor) or colder (next to the AC vent) and that are different form the temperature on the lab bench. Can you find stable microclimates at 20, 22, 24, 26 and 28C?
• Office space sometimes has a different temperature from your lab. (Please use common sense and talk to your safety officer when placing crystallization experiments outside of the lab; i.e. no heavy metal solutions)
• Lab reagent storage fridge or cold room: 4°C. Testing different spots in the fridge may give you a 2C and a 6C spot.
• E.coli incubator or incubation room : 37C, often there's also a 27C or 40C incubator that is used to induce protein expression.

Tip: Measure temperatures several times to identify and avoid spots with undesired temperature fluctuations. Once you've decided on a particular spot: make sure that the crystallization tray is placed inside a container that dampens temperature changes, such as a styrofoam box with an equilibrated cooling element to provide for some extra heat capacity.

All the best,
Peter