(A) Chemical structure of monastrol, a kinesin inhibitor identified in a large-scale screen for small molecules that disrupt mitosis. (B) Normal mitotic spindle seen in an untreated cell. The microtubules are stained green and chromosomes blue. (C) Monopolar spindle that forms in cells treated with monastrol, which inhibits a kinesin protein required for separation of the spindle poles in early mitosis. (B and C, from T.U. Mayer et al., Science 286:971–974, 1999. With permission from AAAS.) example, was identified by this method (Figure 8–20). Chemical inhibitors give the cell biologist great control over the timing of inhibition, as drugs can be rapidly added to or removed from cells, allowing protein function to be switched on or off quickly.
The main technique that has been used to discover the three-dimensional structure of molecules, including proteins, at atomic resolution is x-ray crystallography. X-rays, like light, are a form of electromagnetic radiation, but they have a much shorter wavelength, typically around 0.1 nm (the diameter of a hydrogen 5 µm atom). If a narrow beam of parallel x-rays is directed at a sample of a pure protein, most of the x-rays pass straight through it. A small fraction, however, are scattered by the atoms in the sample. If the sample is a well-ordered crystal, the scattered waves reinforce one another at certain points and appear as diffraction spots when recorded by a suitable detector (Figure 8–21).
The position and intensity of each spot in the x-ray diffraction pattern contain information about the locations of the atoms in the crystal that gave rise to it. Deducing the three-dimensional structure of a large molecule from the diffraction pattern of its crystal is a complex task and was not achieved for a protein molecule until 1960. But in recent years x-ray diffraction analysis has become increasingly automated, and now the slowest step is likely to be the generation of suitable protein crystals. This step requires large amounts of very pure protein and often involves years of trial and error to discover the proper crystallization conditions; the pace has greatly accelerated with the use of recombinant DNA techniques to produce pure proteins and robotic techniques to test large numbers of crystallization conditions.
Analysis of the resulting diffraction pattern produces a complex three-dimensional electron-density map. Interpreting this map—translating its contours into a three-dimensional structure—is a complicated procedure that requires knowledge of the amino acid sequence of the protein. Largely by trial and error, the sequence and the electron-density map are correlated by computer to give the best possible fit. The reliability of the final atomic model depends on the resolution of the original crystallographic data: 0.5 nm resolution might produce a low-resolution map of the polypeptide backbone, whereas a resolution of 0.15 nm allows all of the non-hydrogen atoms in the molecule to be reliably positioned.
A complete atomic model is often too complex to appreciate directly, but simplified versions that show a protein’s essential structural features can be readily derived from it (see Panel 3–2, pp. 142–143). The three-dimensional structures of tens of thousands of different proteins have been determined by x-ray crystallography or by NMR spectroscopy (see page 461)—enough to allow the grouping of common structures into families (Movie 8.1). These structures or protein folds often seem to be more conserved in evolution than are the amino acid sequences that form them (see Figure 3–13).
X-ray crystallographic techniques can also be applied to the study of macromolecular complexes. The method was used, for example, to determine the structure of the ribosome, a large and complex machine made of several RNAs and more than 50 proteins (see Figure 6–62). The determination required the use of a synchrotron, a radiation source that generates x-rays with the intensity needed to analyze the crystals of such large macromolecular complexes.