In this experiment, the structure was determined by X-ray crystallography. To form the AcrB-drug complex, the protein was incubated overnight with either minocycline, doxorubicin, or bromo-minocycline.
X-Ray Crystallography is a method of determining the position of atoms in a large molecule. A large, pure crystal must be made as a sample. X-Rays are directed from a source at the crystal while it is rotating and the electrons in the molecule scatter the X-rays, which can be detected. The scattering patterns are transformed from a 2-d picture to a 3-d one by Fourier transforms and this gives a picture of the electron clouds in the molecule.
Image courtesy of the Lawrence Berkeley National Laboratory Visualization Group, vis.lbl.gov.
The positions of the atoms are then fit to the electron clouds. The crystals must be kept at cryo temperatures so that disorder (vibration of the molecules) is decreased in the crystal and resolution is thus increased. The Phase Problem is solved by soaking the crystals in a heavy metal. In this experiment, the metals used were Mercury, Lead, Platinum, and Selenium.