What is X-ray Crystallography?

In a previous blog, we talked about how newly formed proteins fold into the correct shape. The function of a protein is determined by this folding — but how exactly do researchers determine a protein’s structure? Individual amino acids are far too small to see with normal microscopes, so various specialized methods have been developed to study them. The most commonly used method is X-ray Crystallography, which — despite the recent development of other methods — is still considered to be the gold standard in determining protein structure.

One of the first X-ray diffraction photographs (left) and a modern X-ray diffraction projection (right)

Background

Crystallography, like many disciplines, is full of jargon. We’ll need to introduce a few key terms for this article to make sense:
A crystal is any solid material characterized by a near-perfect repeating arrangement of molecules. Recognizable examples include sugar, snowflakes, and diamonds.
X-rays are a form of electromagnetic radiation initially studied by German physicist Wilhelm Röntgen. With a much shorter wavelength than visible light (0.01 to 10 nanometers), X-rays prove extremely useful when examining materials that are too small to observe with a regular microscope.

A diagram of a simple X-ray diffraction experiment

Diffraction is a phenomenon that occurs when a wave (such as an X-ray) hits an obstacle. Diffraction works through elastic scattering. This is a type of scattering where the wave, interacting with a particle, bounces off in some direction without its wavelength changing.
Crystallographers use X-ray diffraction to produce a pattern on a detector or film. The diffraction pattern is then analyzed to determine the arrangement of the atoms.

History

Crystals were studied scientifically as early as the 17th century, but X-ray crystallography didn’t take off until the early 20th century, when Max von Laue successfully recorded an X-ray diffraction pattern from a copper sulfate crystal. Soon after, Lawrence and William Henry Bragg derived the mathematics to describe this behavior. Bragg’s Law explains the interactions that occur when the atomic spacing in a crystal is comparable to the wavelength of the incident light, and allows us to reconstruct a crystal structure from its diffraction pattern. For their pioneering work, both von Laue and the Braggs won the 1914 and 1915 Nobel Prizes in Physics. In 1952, PhD student Raymond Gosling, working under Rosalind Franklin, took the famous Photograph 51, an X-ray diffraction image of crystallized DNA which was essential in determining its structure. All of these contributions laid the groundwork for the modern discipline of X-ray crystallography.

Photograph 51 was the first X-ray diffraction image of crystallized DNA

The Process

Proteins themselves are not crystals, and many do not crystallize readily. Therefore, protein crystallography is a very involved process. There are many steps necessary to produce a “protein crystal” and determine its structure. First, the protein must be isolated through a series of purification processes that may vary depending on its unique properties. The goal is to generate a homogeneous solution of soluble, properly folded, and stable protein. It may take years to perfect this process for every new protein being crystallized.

Notes from an actual crystallography class at The University of Texas at Austin

Under the right conditions, a supersaturated protein solution will form crystals. Creating a supersaturated solution is easy with small molecules — such as sugar. A common home experiment involves simply boiling water to allow an excess of sugar to be dissolved in it. As the water in this solution evaporates, sugar crystals (“rock candy”) will spontaneously form. With proteins, crystallization is never so simple. Boiled proteins behave more like boiled eggs than boiled sugar — rather than dissolving, they just cook! A common and much more gentle way to achieve protein supersaturation is through vapor diffusion. In this process, the protein sample is placed in a buffered precipitant solution. A drop of this is placed inside a sealed container that houses a reservoir of a more concentrated solution. As water moves to equalize the concentration between the drop and the reservoir, the precipitant and protein concentrations increase in the drop. If this process is sufficiently gradual, well-ordered crystals will develop in the drop.

Using the electron density map and the protein sequence, a computer graphics program can insert each residue into the map and determine its overall structure. Some challenges encountered in this step are small breaks in the continuity of the map, which only become a problem if the path of the protein chain isn’t clear. Another challenge is having a low resolution map, in which case a new or different crystal would have to be analyzed.

The final output is a protein data bank (PDB) file containing atom-coordinates, residue sequence, protein chains, and other relevant information. You can view these PDB files on the Research Collaboratory for Structural Bioinformatics (RCSB) website or download software programs made for viewing them, such as PyMol.

Other Techniques
A few other methods exist for protein structure determination. In fact, the 2017 Nobel Prize in Chemistry was won by the team who developed cryo-electron microscopy. This method involves flash-freezing a protein solution and shining an electron beam through it, to project an image of the electrons’ interactions with the sample. Another technique is nuclear magnetic resonance, where the sample generates a signal characteristic of its nuclei after being perturbed by a weak magnetic field.

X-ray crystallography has greatly improved our understanding of the macromolecular world and is important to the advancement of many biosciences. It is essential to the biotech industry, Macromoltek included, for novel drug and enzyme design.

Links and Citations:
1. Smyth MS, Martin JHJ. x Ray crystallography. Molecular Pathology. 2000;53(1):8–14.
2. Lawson, Dave. A Brief Introduction to Protein Crystallography. https://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htm
3. X-ray Crystallography Platform. https://www.creative-biostructure.com/x-ray-crystallography-platform_60.htm
4. 65 years on from Photo 51 https://alumni.kcl.ac.uk/news-features/65-years-on-from-photo-51
5. Wellcome Collection. https://wellcomecollection.org/works/y2d2nar6

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