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The University of Southampton
Structural Biology Part of Biological Sciences

X-ray Crystallography

Proteins or nucleic acids can form crystals suitable for X-ray diffraction experiments. These ultimately allow the determination of the atomic structure of the macromolecule. This information helps us understand how a drug molecule binds to its macromolecular target or how macromolecules interact, for example in signal transduction pathways or in the molecular recognition events that occur in the immune system. More generally the technique can tell us how macromolecules are shaped to perform their biological function.

Protein or DNA sample preparation is key to success in crystallisation and structure determination. Crystallisation conditions are sample specific and hundreds of combinations of buffers, salts, organic polymers, metal ions and additives are typically screened. The results can be beautiful as seen in the video below, but they are also scientifically very exciting as they allow us to understand how the biomolecules work.

Typically, highly pure protein or DNA would enter the crystallography pipeline at a concentration of 0.2mM or higher. About 50μL is enough to test the 96 conditions that form the entry screen. The buffer the sample should be supplied in will depend on the stability of the sample but 20mM HEPES pH 7.5, 100mM NaCl is a good starting point. The screens used for crystallisation use buffering components at 100mM so it's important that the sample buffer does not compete with this.

Once the crystal plate is prepared, it's incubated at 4°C or 20°C and imaged under a microscope either manually or using one of the dedicated imagers which images all crystal wells and matches the pictures to the protein and screen conditions in software called CrystalTrak.

Any crystals appearing are UV imaged to confirm they are biomolecules and not salt before being frozen in liquid nitrogen and stored for X-ray diffraction.

Data collection and processing is carried out at Southampton. National and international synchrotron facilities are routinely used to gain higher resolution data or to use microfocus sources that can target the highest diffracting region within a single crystal.

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