Experimental methods: Advantages and disadvantages
The primary and most direct experimental methods used in the study of tertiary protein structure include:
◦ Protein crystallography
◦ Electron microscopy (Cryo-EM): Electron crystallography and single particle reconstruction
◦ Nuclear magnetic resonance (NMR spectroscopy)
◦ Small-angle X-ray and neutron scattering (SAXS and SANS)
◦ Homology modelling
As always, each experimental method has its advantages and limitations. Thus, electron microscopy, and particularly Cryo-EM, which provides higher resolution, can be used to study relatively large objects, like cellular organelles or large macromolecular complexes, like in the case of single-particle reconstruction. An advantage of the method of single-particle reconstruction, in comparison to protein crystallography, is that it does not require the protein to be crystallized, since crystallization in many cases may be difficult and may require a large amount of effort. However, in electron crystallography, which is primarily used for membrane proteins, we need two-dimensional crystals of a protein.
Cryo-EM also requires small amounts of material, which is an advantage in comparison to both crystallography and NMR spectroscopy. NMR requires much larger amounts of material and in addition, the protein in question needs to be stable at room temperature under a rather long time of data acquisition. One of the limitations of Cryo-EM is that the resolution obtained is generally limited in comparison to the resolution obtained from NMR spectroscopy or protein
A schematic picture showing MAX IV, the future synchrotron radiation facility which will be built in Lund, Sweden
Cryo-EM also requires small amounts of material, which is an advantage in comparison to both crystallography and NMR spectroscopy. NMR requires much larger amounts of material and in addition, the protein in question needs to be stable at room temperature under a rather long time of data acquisition. One of the limitations of Cryo-EM is that the resolution obtained is generally limited in comparison to the resolution obtained from NMR spectroscopy or protein crystallography. Protein crystallography, on the other hand, may provide atomic or near atomic resolution, when small details of the protein structure can be resolved with very high accuracy. Although NMR spectroscopy does not provide this level of resolution, the method proved to be valuable when details of the dynamics of the system needed to be studied. SAXS and SANS measurements are performed in solution, thus having the advantages of Cryo-EM and NMR. However, as in Cryo-EM, the resolution obtained from these methods is limited and in addition, the requirement for the homogeneity of the protein sample is high.
Homology modeling may also be used for obtaining three-dimensional information on protein structure. However, for accurate modeling we need high percentage identity between the amino acid sequence of the given protein and its homologue for which the tertiary structure is known (the template). Additional methods used in obtaining partial (local) structural information include mass spectrometry, analytical ultracentrifugation, various fluorescent spectroscopic methods, etc.
In the following chapters I will discuss the basic ideas of protein X-ray crystallography, Cryo-EM and SAXS and provide more details of the experiment procedures.
Homology modeling may also be used for obtaining three-dimensional information on protein structure. However, for accurate modeling we need high percentage identity between the amino acid sequence of the given protein and its homologue for which the tertiary structure is known (the template). Additional methods used in obtaining partial (local) structural information include mass spectrometry, analytical ultracentrifugation, various fluorescent spectroscopic methods, etc.
In the following chapters I will discuss the basic ideas of protein X-ray crystallography, Cryo-EM and SAXS and provide more details of the experiment procedures.