Structural computational biology: introduction and background  (Page 3/6)

Unlike other structure determination methods, with x-ray crystallography, there is no fundamental limit on the size of the molecule or complex to be studied. However, in order for the method to work, a pure, crystalline sample of the protein must be obtained. For many proteins, including many membrane-bound receptors, this is not possible. In addition,a single x-ray diffraction experiment provides only static information - that is, it provides only information about the native structure of the protein under the particular experimental conditions used. As we will see later, proteins are often flexible, dynamic objects when in their natural state in solution, so a single structure, while useful, may not tell the full story. More information on X-ray Crystallography is available at Crystallography 101 and in the Wikipedia .

Nmr

Nuclear Magnetic Resonance (NMR) spectroscopy has recently come into its own as a protein structure determination method. In an NMR experiment, a very strong magnetic field is transiently applied to a sample of the protein being studied, forcing any magnetic atomic nuclei into alignment. The signal given off by a nucleus as it returns to an unaligned state is characteristic of its chemical environment. Information about the atoms within two chemical bonds of the resonating nucleus can be deduced, and, more importantly, information about which atoms are spatially near each other can also be found. The latter information leads to a large system of distance constraints between the atoms of the protein, which can then be solved to find a three-dimensional structure. Resolution of NMR structures is variable and depends strongly on the flexibility of the protein. Because NMR is performed on proteins in solution, they are free to undergo spatial rearrangements, so for flexible parts of the protein, there may be many more than one detectable structures. In fact, NMR structures are generally reported as ensembles of 20-50 distinct structures. This makes NMR the only structure determination technique suited to elucidating the behavior of intrinsically unstructured proteins , that is, proteins that lack a well-defined tertiary structure. The reported ensemble may also provide insight into the dynamics of the protein, that is, the ways in which it tends to move.

NMR structure determination is generally limited to proteins smaller than 25-30 kilodaltons (kDa), because the signals from different atoms start to overlap and become difficult to resolve in that range. Additionally, the proteins must be soluble in concentrations of 0.2-0.5 mM without aggregation or precipitation.For more information on how NMR is used to find molecular structures, please see NMR Basics and The World of NMR: Magnets, Radio Waves, and Detective Work at the National Institutes of Health's The Structures of Life website.

Electron diffraction

Electron diffraction works under the same principle as x-ray crystallography, but instead of x-rays, electrons are used to probe the structure. Because of difficulties in obtaining and interpreting electron diffraction data, it is rarely used for protein structure determination. Nevertheless, ED structures do exist in the PDB. For more on ED, see this Wikipedia article .