However, even at cryogenic temperatures, radiation damage may limit the final resolution of the structure ( 6). Finding appropriate freezing conditions to minimize radiation damage can be a further challenge for membrane protein structure determination. Due to the large problem of radiation damage, data collection from protein crystals is currently done nearly exclusively at liquid-nitrogen temperature. Even if these obstacles are overcome, the growth of large well-ordered single crystals is the next hurdle and bottleneck for membrane protein crystallography. There are several challenges for membrane protein crystallography, which involve overexpression, purification, and stabilization of the proteins. 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. The techniques are complementary and have contributed significantly to the understanding of the structure and function of proteins, and of membrane proteins in particular. 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). 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 ( 1–3), human aquaporin-5 ( 4), and human leukotriene C 4 synthase ( 5). However, structural information is rare for large multiprotein complexes and membrane proteins, with 60% of all drugs currently available targeted at membrane proteins. To date, >60,000 protein structures have been solved by x-ray crystallography, electron microscopy, and NMR. 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. Therefore, structure determination is one important clue to the complexity seen in biological macromolecules. The structure and function of molecules are strongly related at the atomic and molecular levels. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Data were collected from crystals ranging in size from 100 nm to 2 μm. ![]() As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |