Crystallization

A wide range of protein crystallization tools is available. As a standard procedure, the first crystallization trials are done with one of our crystallization screens at room temperature or at 4 ^oC using a vapor diffusion sitting-drop method in 96-well crystallization plate or using a crystallization under oil method in 96-well Douglas Microbatch plate. The standard drop volumes are 1–2 μL for sitting drops and 0.5 μL for Microbatch under oil.

In the case of crystals or “promising” precipitate the crystallization conditions are optimized varying the pH and the precipitant concentration around the condition of the hit. The optimization is carried out using the CrysScreen software of the Tecan robotics system using the sitting-drop vapor diffusion method in 96-well plates. If a suitable crystallization condition has been found, it is also worthwhile to optimize the condition using the hanging drop (or sitting-drop) vapor diffusion method in 24-well plates and bigger drops (5 μL + 5 μL) from which the crystal mounting is easier and the crystals tend to grow to a larger size. Macro and micro seeding methods are also used.

Tools for protein crystallization in very small capillaries (less than 0.2 mm in diameter) for diffusion controlled mixing, avoiding convection, are also available. A recent description can be found in the second edition of Protein Crystallization (Bergfors, 2009).

Figure 1.Schematic presentation of the current protein crysatllization setup.

Sample requirements

Protein sample intended for crystallization has to be pure and preferably not in phosphate buffer and not in the presence of any additional salt. Protein concentration should be approximately 5 mg/ml and the buffer concentration should be around 25 mM. The minimum required quantity is 120 μL (0.6 mg protein in total).

Pipetting robots

The protein crystallization is centered around the Tecan pipetting robot (Figure 2 on the left) which is used for the preparation of crystallization screens and for setting up crystallization experiments. The robot has a very flexible software by which way different crystallization set-ups can be handled. More recently a robot for the crystallization of membrane proteins has been installed (Figure 2 in the middle).

Facilities

There is a temperature controlled laboratary (at 20 ^oC) as well as a cold room dedicated for crystal growth and crystallization experiment storage. There are three microscopes for inspection of crystallization drops and for crystal mounting and handling, of which one is in the cold room and one is equipped with a CCD camera connected to a PC for documentation of the crystallization results. In addition, we have two programmable personal crystallization boxes (PCB), which are used to explore and utilize the temperature-dependency of the crystallization process (Figure 2 on the right).

Figure 2.On the left the Tecan pipetting robot which is used for the preparation of the crystallization solutions and for setting up crystallization experiments. In the middle the pipetting robot for crystallization of membane proteins. On the right the personal crystallization boxes which can be programmed to a certain temperature or to generate a temperature gradient.

Crystallization screens

Stock solutions are readily available for several different in-house screens as well as for some commercial screens, stored in deep well blocks. These screens, resulting in approximately 500 starting conditions, are documented on our wiki which is accessible to local users via the X-ray homepage. Currently, we are using the following screens for the initial screening:

• Factorial screen 1&2
  
• Clear Strategy Screen I&II
• PEG Smear
• Crystal Screen I&II (Hampton)
• ProPlex (Molecular dimensions)

Factorial 1 screen was developed to test a broad range of buffers, precipitants and additives using only a small amount of protein (Zeelen et al., 1994). It is based on the sparse-matrix screening method by Jancarik and Kim (1991) and the crystallization conditions are selected from the Biological Macromolecule Crystallization Database for crystals of over 600 macromolecules (Gilliland GL, 1988). Factorial 2 screen is an additive screen to test several solvents, salts and ions affecting to protein solubility. Currently, these two screens are combined into a single screen consisting of 96 crystallization conditions.

Clear strategy screen was developed by Brzozowski and Walton (2001) at University of York especially for the crystallization of enzymes. The screen allows to choose the pH according to the known properties of a particular protein such as isoelectric point, solubility or stability. The screen utilizes precipitants which can be used as a cryoprotectants and which provide a potential anomalous scatterer. In our home made screen this clear strategy screen is combined with sodium malonate (McPherson, 2001) and ammonium sulphate screens.

Polyethylene glycols (PEG) are common precipitants used for protein crystallization. PEG-Smear screen is a home made screen which utilizes the idea of Janet Newman (2005) to use a mixture of different molecular-weight PEGs for screening purposes.

Crystallization plates

Crystallization experiments are set-up into a 96-well or a 24-well crystallization plates depending on the experiment (screening, optimization, soaking, hanging drop, sitting drop). For the sitting drop vapor diffusion set-ups we are using the Corning 3556 plate and the Greiner 609101 CrystalQuick Crystallization plate. The Corning plate is used for the initial screening and the sitting drops can be set-up using either the Tecan pipetting robot or manually using the 8-channeled pipette. The Greiner plate has 3 drop wells giving an option to screen three different proteins, protein concentration or ligands at the same time. The drops are prepared manually using a home made plate cover to decrease the evaporation while preparing the drops. The plates are covered with a sealing tape.

For the hanging drop vapor diffusion setup we are using the PVC plates with 12 wells (Zeelen plates), two such plates are placed into a sandwich box for easy handling of the crystallization experiments. The drops are set-up manually on the siliconized cover glasses and grease is applied between the cover glass and the plate to seal the well.

The microbatch under oil screen, where a small volume of protein solution (0.5 μL) and the crystallization solution (0.5 μL) are pipetted underneath an oil layer, are carried out using the Douglas microbatch plate using the Tecan robot.

Additional information

There is much more information available for the local users, on the protein crystallography wiki, accessible to local users via the X-ray homepage.

References

Bergfors TM (editor) Protein crystallization, 2nd edition, International University Line (2009), USA.

Brzozowski A M & Walton J (2001) Clear strategy screens for macromolecular crystallization. J. Appl. Cryst. 34:97-101.

Gilliland GL (1988) A biological macromolecule crystallization database: A basis for a crystallization strategy, J. Cryst. Growth 90:51-59.

Jancarik J & Kim S-H (1991) Sparse-matrix sampling: a screening method for crystallization of proteins. J. Appl. Cryst. 24:409-411.

McPherson A (2001) A comparison of salts for the crystallization of macromolecules. Protein Sci. 10:418-422.

Newman J, Egan D, Walter TS, Meged R, Berry I, Ben Jelloul M, Sussman JL, Stuart DI, Perrakis A (2005) Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta Cryst. D 61:1426-1431.

Zeelen JP, Hiltunen JK, Ceska TA, Wierenga RK (1994) Crystallization experiments with 2-enoyl-CoA hydratase, using an automated 'fast-screening' crystallization protocol. Acta Cryst D 50:443-447.

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Updated August 19th, 2011