Experimental Methods in Structural Biology

This chapter's primary focus is on the basics of the most powerful experimental method used in protein structure determination, X-ray crystallography. Crystallography has significantly contributed to understanding the structure of matter at the level of atoms and molecules, including the structure of proteins, nucleic acids, and other biological macromolecules. The history of crystallography starts from the discovery of X-ray diffraction by crystals by Max von Laue, which resulted in the first Nobel Prize awarded to this field of science in 1914. This was followed by the work of William Henry Bragg and William Lawrence Bragg (father and son) "for their services in the analysis of crystal structure using X-rays" (Nobel Prize in Physics, 1915). The Brags laid the foundation of X-ray crystallography. Since then, many Nobel prizes have been awarded for significant achievements in structural biology. I will try to review this at some moment in the future.

Throughout the years, other experimental techniques have been adopted to study the structure and function of biological macromolecules. The most important of those are:

  • Nuclear magnetic resonance (NMR spectroscopy)
  • Cryo-Electron microscopy
  • Small-angle X-ray and neutron scattering (SAXS and SANS)
Each experimental method has its advantages and limitations. Thus, electron microscopy, particularly Cryo-EM, can provide high resolution structure and be used to study objects like cellular organelles, proteins, and macromolecular complexes, which are difficult to crystallize. Cryo-EM requires small amounts of material, which is an advantage compared to crystallography and NMR spectroscopy. Cryo-EM has also received its share of Nobel prizes. In 2017 the Nobel Prize was to Jacques Dubochet, Joachim Frank & Richard Henderson 2017 “for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.”

NMR requires much more significant amounts of material, and in addition, the protein under study needs to be stable at room temperature during data acquisition. Like crystallography and Cryo-EM, NMR spectroscopy techniques have also received several Nobel Prizes. Thus, the prizes were awarded to Felix Bloch and Edward Purcell in 1952, Richard Ernst in 1991 & Kurt Wüthrich in 2002. Kurt Wüthrich shared the award with John B. Fenn and Koichi Tanaka. They contributed to the development of mass spectrometry, which is also widely used in studying macromolecules and macromolecular complexes. Interesting to look at the motivation for the prize, as stated by the Nobel committee:

"for the development of methods for identification and structure analyses of biological macromolecule ionizations," with one half jointly to John B. Fenn and Koichi Tanaka "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules" and the other half to Kurt Wüthrich "for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution."

SAXS and SANS are also excellent methods for studying protein structure, although they provide limited resolution. An advantage of these methods is the ability to analyze large complexes directly in solution, allowing better control of the experimental conditions. In addition, the dynamic behavior of large macromolecular complexes and their oligomeric states in the solution can be studied. An example of using SAXS is a study (in which I was involved) that targeted the oligomerization state in a solution of a protein called frataxin. The protein forms different oligomeric states (dimers, trimers, and higher-order oligomers) in response to higher concentrations of iron in the solution. We were able to follow the process of oligomerization and separate the different oligomeric states using SAXS (published in Söderberg et al., 2011)

An outline of X-ray crystallography
Crystallography starts with a crystal, and the protein must first be crystallized to get crystals! Difficulties associated with crystallization are probably the major limitation of X-ray crystallography. People often say: Crystallization is an art and not a science. However, some general principles still need to be followed, and most importantly, different methods have been developed to facilitate protein crystallization. After obtaining crystals, it is time for the X-ray crystallographic experiment to place the crystal in an X-ray beam, rotate it, and collect diffraction data. Once data have been obtained, the rest of the work will be done using specialized software packages.

I will first give an overview of the method of crystallization and crystallization tools, followed by an overview of crystallography, X-ray data collection, refinement, and structure quality:

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