Proteins play a crucial role in the world of research and their structures are, therefore, of great interest to the researcher.
Many techniques and methods have been used to determine this, but one of the most important has been the discovery and development of X-ray crystallography.
The tertiary structure of a protein is responsible for its properties and behavior. The primary methods in use to study this include:
X-Ray Crystallography
This method provides the maximum data regarding the tertiary structure of a protein. X-rays are passed through the rotating crystal and the diffracted rays are collected on a target and analyzed by computerized systems.
Crystal quality is crucial; thus it results in very detailed data on atomic arrangement within a rigid protein with other neighboring ions, ligands and molecules, but may not work so well with proteins which have large flexible domains and therefore do not form precisely ordered crystalline structures. It requires relatively large amounts of the protein.
For this reason recombinant proteins are often produced in the necessary quantity, and then impurities removed, followed by refolding of the protein and crystallographic study. It provides very high resolution.
Understanding Crystallography - Part 1: From Proteins to Crystals Play
Electron Microscopy and Cryo-Electron Microscopy
These methods are suited to the study of large macromolecule complexes or even cellular organelles, which are relatively bigger on the molecular scale, and can help to reconstruct the tertiary structure of a single particle. This has the great advantage of obviating the challenging prerequisite of protein crystallization.
Cryoelectron microscopy is a variant at temperatures at or below that of liquid nitrogen and can visualize protein structures at very high resolution, though is less than that of methods like NMR spectroscopy or crystallography.
It works with minute amounts of protein as well and reduces the artefacts due to radiation damage.
What is Cryo-Electron Microscopy (Cryo-EM)? Play
Nuclear Magnetic Resonance (NMR) Spectroscopy
This method depends on the effect of varying radiofrequency waves on nuclear resonance of various atoms within the protein.
It needs larger quantities of protein in a stable soluble form at room temperature, and to remain stable for the long duration of data acquisition.
The proteins should be of small size as well to avoid overlapping peaks.
However, it gives a higher resolution. It is most suitable when protein crystallization is not feasible as with flexible proteins, or when system dynamics are to be detailed.
Small-Angle X-Ray Scattering and Small-Angle Neutron Scattering
These methods are valuable for studying protein structure when limited resolution is sufficient. Experimental conditions can be better controlled because the protein is usually in solution.
Homology Modeling
This enables researchers to obtain a three-dimensional picture of a protein. It needs prior knowledge of the close homologue or template which has a very high degree of identical amino acid sequence to the analyte.
Protein modeling based on this template depends on the fact that protein structure is highly conserved in most cases.
Partial Structural Study Methods
These include ultracentrifugation, mass spectrometry and fluorescence spectrometry, which may be used to complement other techniques.
References
https://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Protein_Structure_determination_methods http://www.proteinstructures.com/Experimental/experimental-methods.html https://pdb101.rcsb.org/learn/guide-to-understanding-pdb-data/methods-for-determining-structure
Further Reading