X-Ray Crystallography Lab
The Sacchettini lab uses several techniques in order to evaluate the unseen world of proteins and other molecules. These techniques range from X-Ray crystallography to High throughput screening.
X-ray crystallography is a method of seeing the structure of molecules that can’t be seen with a microscope. To do this, first the molecule of interest, such as a protein, must be arranged in crystal form. This is accomplished by putting the protein into a solution that encourages the molecules to align themselves in an ordered fashion. This solution usually has a high salt or polymer concentration and often is pH regulated to keep the protein folded in a physiologically relevant way. Care must be taken not to over concentrate this solution or the protein will precipitate out of solution in a disordered form which can not be analyzed. The crystallization step is often the most time consuming and some proteins are not amenable to crystallization, making this the rate limiting step in crystallography.
Next, the crystal is mounted in an x-ray diffractometer, a machine that can shoot a narrow band of x-rays through the crystal and catch their change in location as they come out the other side. The x-rays pass through the crystal until they hit an atom, or more accurately, the electric field generated by the atom's electrons. The electric field deflects (diffracts) the x-rays in a specific manner.
Because in a crystal the molecules are arranged in a regular pattern the light waves can constructively interfere and enhance the signal of the diffracted x-rays sufficiently for detection. The reflections are captured and transferred to a computer.
Because proteins are three dimensional, a single two dimensional image cannot give sufficient information to reconstruct what the entire protein looks like, just as a photograph of part of a person’s head cannot tell you how they look when viewed from all other angles. For this reason, images of the protein crystal from all angles are needed to get a complete picture. Once all images have been taken a computer assembles the spots from all the images and pieces together a model of the three dimensional protein. Because the x-rays have been deflected by the electron fields of the atoms the result is a picture of where the electrons are in the protein
By using the shape of the electron density and the known amino acid sequence of the protein, it is possible to create an accurate model of the position of each atom and bond within the protein. The final protein model can be presented in a variety of ways. The nature of a crystal requires that the shape of the molecules within it be identical. This presents a problem when determining the structure of a protein that experiences dramatic conformational change as part of its function. The crystal structure obtained is a snapshot of the protein at one point and in order to obtain a more complete picture of a protein many different crystals of the same protein may be grown in order to obtain a model of different conformations of the same protein.