Protein Crystallization for X-ray Crystallography

Summary

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Abstract

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.

To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).

Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.

Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

Protocol

Materials: Protein sample – Lysozyme (50 mg/ mL) Hanging drop 24-well tray Sitting drop 24-well tray Microbatch crystallization 96 well tray Crystallization solutions (either commercial available or home made) Silicon grease 5 mL syringe without Luer-lock Siliconized cover slides Optical sealing tape Paraffin 0.1-2 μL micropipette with low-retention tips Tweezers Professional wipes <p clas…

Representative Results

Crystallization is usually referred to as the bottleneck of X-ray crystallography. A sparse matrix incomplete factorial screen of precipitating conditions typically produces many different types of protein aggregation and precipitation, among them large single crystals. If the protein or precipitant concentrations are too high one can see brown matter with no distinct shape and size (amorphous precipitation). When the solution is undersaturated, the drop will often be completely clear and devoid of any kind of precipitat…

Discussion

In this article we describe and demonstrate general current protocols for protein crystallization. Since it a multi-step procedure there are few considerations one needs to be aware of. When working with very small volumes (0.5-2 μL), drying of the drop due to evaporation is a major concern. Hence, it is recommended to work in a well-controlled environment (with low air flow, high humidity and tight temperature control) and to adopt a technique that minimizes exposure of the drop to the open air. For this reason, it …

Materials

New Item
Lysozyme		Sigma-aldrich	L6876-1G
24 well VDX Plate		Hampton research	HR3-142
24 well Cryschem Plate		Hampton research	HR3-158
Dow Corning Vacuum Grease		Hampton research	HR3-510
Siliconized glass circle coverslides		Hampton research	HR3-231
100% paraffin oil		Hampton research	HR3-411
1.88 inch wide Crystal Clear Sealing Tape		Hampton research	HR3-511
96 Well Imp@ct Plate (Microbatch plate)		Hampton research	HR3-098