What is X-Ray Crystallography?
X-ray crystallography is the most important method for determining protein structure. X-ray crystallography is a technique by which an x-ray beam is scattered upon contact with a crystallized protein. The pattern of scattering can be used to determine the relative position of the atoms in the crystal.
The quality of an x-ray crystal structure is described in terms of its resolution. Resolution is listed in units of angstroms (Å). A high resolution for an x-ray structure might be 0.5–2.0 Å with a lower number indicating better resolution.
Once the data has been processed, the positions of nonhydrogen atoms appear as spheres in three-dimensional space in an image called an electron density map. The electron density map shows atoms as spheres.
Figure 9.1 shows the impact of resolution on the readability of an electron density map. Image (a) has a resolution of 0.62 Å. Atoms are nearly distinct and separate from other atoms. Image (b) has a resolution of 1.2 Å. Atoms are still clear but somewhat larger.
As the resolution drops to 2.0 Å (c), the spheres of the individual nuclei become unclear. The feature that now dominates the electron density map is the peptide backbone. Once the resolution drops to 3.0 Å (d) and 4.0 Å (e), single atom branches off the backbone are nearly imperceptible. At 5.0 Å (f), folds of the backbone can mistakenly appear to merge together.
Solving an x-ray structure requires correctly assigning atoms in the protein to match the electron density map. Before solving a structure, an x-ray crystallographer already has the sequence of the target protein from the molecular biology team.
The x-ray crystallographer begins to solve the structure by aligning the most obvious residues, such as the aromatic rings, into the electron density map. Less obvious residues then fall into place as possibilities become more limited. If an electron density map has a low resolution (6–8 Å), then only general features such as a-helices or b-sheets may be clear. The confident placement of individual residues is not possible.
A protein will generally have one or more random coil regions in its overall structure. Random coils, as the name suggests, do not have a regular, folded structure and undergo rapid, continuous conformational changes. These regions appear indistinct even in an otherwise high resolution x-ray structure. Fortunately, binding and active sites generally occupy ordered protein regions that are amenable to characterization by x-ray crystallography.
Knowing the structure and shape of the binding site of a target allows the discovery team to make informed decisions about molecules that will bind to the target. Is the binding site deep or shallow? Are the amino acid residues in the binding site polar, nonpolar, or charged?
The properties of the binding site determine the optimal, complementary properties needed in a drug that will interact with the binding site. Often molecules can be cocrystallized with an enzyme target. The x-ray structure of the complex shows both how a compound fills the binding pocket and precisely where a compound may be modified to better fill the active site.
No comments:
Post a Comment