We all are familiar with crystals from rock collections or small molecules, such as salt or sugar. We usually associate them with properties like hard, durable, and pretty. Unfortunately, only the latter is true for protein crystals.
Proteins consist of long macromolecule chains made up from 20 different amino acids. The chains can be several hundred residues long and fold into a 3-dimensional structure. It is therefore quite understandable that protein molecules have irregular shapes and are not ideally suited to be stacked into a periodic lattice, i.e., a crystal. Protein crystals are thus very fragile, soft (think of a cube of jelly instead of a brick) and sensitive to all kind of environmental variations. Protein crystals contain on average 50% solvent, mostly in large channels between the stacked molecules on the crystal. The interactions holding the molecules together are usually weak, hydrogen binds, salt bridges, and hydrophobic interactions, compared to strong covalent or ionic interactions in mineral crystals. This explains the fragility of the crystals, but allows for the possibility of soaking metal solutions (important for phasing) or even large enzyme substrates or inhibitors, into the crystals.
You can read more about protein structure in the first pages 1, 2, 3 of Chapter 2 of my book Biomolecular Crystallography or buy the book from Amazon.
The Experimental Setup
In order to obtain a crystal, the protein molecules must assemble into a periodic lattice. One starts with a solution of the protein with a fairly high concentration (2-50 mg/ml) and adds reagents that reduce the solubility close to spontaneous precipitation. By slow further concentration, and under conditions suitable for the formation of a few nucleation sites, small crystals may start to grow. Often very many conditions have to be tried to succeed. This is usually done by initial screening, followed by a systematic optimization of conditions. Crystal size should to be from a few hundred down to about 20 micron in each direction to be useful for diffraction experiments.
The most common setup to grow protein crystals is by the hanging drop technique : A few microliters of protein solution are mixed with an about equal amount of reservoir solution containing the precipitants. A drop of this mixture is put on a glass slide which covers the reservoir. As the protein/precipitant mixture in the drop is less concentrated than the reservoir solution (remember: we mixed the protein solution with the reservior solution about 1:1), water evaporates from the drop into the reservoir. As a result the concentration of both protein and precipitant in the drop slowly increases, and crystals may form. There is a variety of other techniques available such as sitting drops, dialysis buttons, and gel and microbatch techniques.
Robots are commonly used for automatic screening and optimization of crystallization conditions. There are many useful kits for crystallization screening. An inherently efficient random screen for crystallization conditions is CRYSTOOL. The main advantage is the small sample size a crystallization robot can handle reproducibly, but it needs some effort to set it up. Click here to learn more about the IMPAX robot for batch crystallization or the multi-channel PHOENIX for 96 well sitting drop plates.
Left: 24 basic hanging drop experiments are set up manually in a Linbro plate. Center right : A kit of different screening solutions, a set-up Linbro plate, dialysis buttons and a micro batch plate behind a goniometer head. Right: a PHOENIX robot can set up 96 different crystallization experiments in sitting drop format in about 2 minutes.
You can read more about protein crystallization in the first pages 1, 2, 3 of Chapter 3 of my book Biomolecular Crystallography or buy the book from Amazon.
There are some crystal growth videos on my site.
Physical chemistry of crystallization
To learn more about the theory behind phase relations used in protein crystallization pathway diagrams, got to my EMBL lecture notes (this is heavy stuff, easier explained in my book).
To read more about high throughput considerations, got to my EMBL lecture notes (better and updated content in my book).
To learn more about tricks and supplies for crystal growing click here
Helpful techniques to screen proteins
The chances for success in crystallizaion experiments depend strongly on the conformational purity of a protein. A single band on a denaturing SDS gel is a good sign, but Dynamic Light Scattering and Circular Dichroism help to learn about the aggregation and dispersity or the folding of your protein sample. Follow the links for more.
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This World Wide Web site conceived and maintained by Bernhard Rupp. Last revised May 10, 2010 16:02