X-ray Diffraction

Garrus_Vakarian · September 22, 2014

The structure and function of molecules are strongly related at the atomic and molecular levels. Therefore, structure determination is one important clue to the complexity seen in biological macromolecules. The structure forms the basis for elucidation of the reaction mechanisms and understanding how the structure relates to the function and the dynamics of the molecules. To date, >60,000 protein structures have been solved by x-ray crystallography, electron microscopy, and NMR. However, structural information is rare for large multiprotein complexes and membrane proteins, with <300 unique membrane protein structures determined to date. Membrane protein structure determination is of extreme importance for understanding fundamental principles in biology, because these proteins are the key players in the most important processes of all living cells, such as respiration; photosynthesis; ion, nutrient, and hormone transport; cell communication; signal transduction; vision; and nerve function. About 30% of all proteins in cells are membrane proteins, and they are of great importance for human health, with >60% of all drugs currently available targeted at membrane proteins. Despite their extremely high impact, only three medically relevant human membrane protein structures have been determined to date, that of a G-protein-coupled receptor (1, 2, 3), human aquaporin-5 (4), and human leukotriene C₄ synthase (5).

Three major techniques have been established to date for structure determination of proteins: x-ray crystallography, NMR, and electron microscopy (single-particle and electron crystallography). The techniques are complementary and have contributed significantly to the understanding of the structure and function of proteins, and of membrane proteins in particular. The majority of protein structures in the Protein Data Bank have been determined by x-ray crystallography, which requires the growth of large, well-ordered protein crystals. There are several challenges for membrane protein crystallography, which involve overexpression, purification, and stabilization of the proteins. Even if these obstacles are overcome, the growth of large well-ordered single crystals is the next hurdle and bottleneck for membrane protein crystallography. Due to the large problem of radiation damage, data collection from protein crystals is currently done nearly exclusively at liquid-nitrogen temperature. Finding appropriate freezing conditions to minimize radiation damage can be a further challenge for membrane protein structure determination. However, even at cryogenic temperatures, radiation damage may limit the final resolution of the structure (6).

The size of the crystals used for x-ray crystallography is of particular importance, because the integrated intensity of an x-ray diffraction peak from a crystal is proportional to the ratio of its diffracting volume to its unit cell volume. The size of a typical single crystal used for conventional protein crystallography would be on the order of 50–500 μm. The difficulties associated with the crystallization of proteins are well known (6, 7), and the larger and more complicated the protein structure becomes, the more challenging are the tasks required to isolate the protein intact and to grow large, well-diffracting crystals. In particular, membrane proteins are notoriously difficult to crystallize due to their amphiphilic nature (8).

An example of a difficult-to-crystallize membrane protein is Photosystem I (PSI), which has been used as a test protein for our studies. Photosystem I is a large membrane protein complex that catalyzes the second light-induced charge separation step of oxygenic photosynthesis. In cyanobacteria, the protein is a trimer with a molecular mass of 1,056,000 Da. The first micron-sized crystals of PSI, from the thermophilic cyanobacteriumThermosynechococcus elongatus, were reported in 1988 (9). The first structural model of PSI, based on crystal diffraction to 6 Å resolution, was determined in 1993 (10, 11), followed by a 4-Å structure in 1996 (12) and an improved structure at 4 Å in 1999 (13,14). All of these medium-resolution structures were solved from large crystals that were shifted after each diffraction-pattern recording during the data collection, as it was extremely difficult to establish freezing conditions due to the weak crystal contacts and the high solvent content of 78%. In 2001, the structure of Photosystem I was unraveled at 2.5 Å resolution (15) from crystals that were incubated in sucrose before freezing. Thus, 13 years intervened between the growth of the first microcrystals and determination of the first near-atomic-resolution structure of Photosystem I based on large, well-ordered single crystals under cryogenic conditions (16).