Biochem 660 - 2008 Molecular)Graphics)Essentials)15 Desktop Molecular Graphics Background Essentials 1 Introdu ction) The visualization techniques of the structure of macromolecules are companion tools to the sequence analysis algorithms. New DNA sequences are being cloned and sequenced rapidly but the structure of the putative encoded proteins cannot be determined based only on their sequence. As the number of protein structures solved by x-ray crystallography is increasing, it will become easier to find structural homologs to fit onto newly protein sequences. Molecular graphics play a key role in understanding current structures and creating (structural) models. Molecular graphics have evolved over the last 40 years from a simple vector display on a high performance oscilloscope to sensor-based virtual reality. For a beautifully illustrated account of “History of Visualization of Biological Macromolecules” see http://www.umass.edu/microbio/rasmol/history.htm. Image reference: http://www.umass.edu/molvis/francoeur/levinthal/lev-index.html Desktop computers are now more powerful than mainframes of the last decades and there are free and commercial desktop software to manipulate 3 dimensional structures for the creation of publication quality images to illustrate research papers, proposals and to help visualize target molecules, their structural properties or their interaction with other molecules or ligands. To be able to manipulate 3 dimensional structures on a desktop computer with a molecular graphics software is critical for today's molecular biologist and a necessary complement to sequence analysis projects. 2 Where) do)3)dimensional)structures)c ome)from?)) In summary there are three main methods: X-ray crystallography, NMR and 3D image reconstruction from cryo-electron microscopy. Biochemists and crystallographers have developed techniques to crystallize macromolecules. Indeed proteins, nucleic acids or their complex can form crystals in specific biochemical conditions. The crystals are very fragile and small (often less than a millimeter) but they still can be placed inside an x-ray beam. Because of the regular16 Molecular)Graphics)Essentials) arrangement of the molecules within the crystals the x-ray will diffract in a very specific pattern that can be recorded on x-ray photographic film or an electronic array detector. With the help of powerful computer and complex software, the mathematical analysis of the diffraction pattern allows the crystallographer to calculate where the electrons (of the atoms) of the protein would be located in 3D space inside the crystal. They then fit a wireframe representation of the amino acids inside the electron density. When the position of the atoms is refined, the structure is published and usually deposited at the Protein Data Bank. These are the structures that you can fetch with a web browser and display inside on your desktop computer. A notable exception is for structures determined in the private sector these coordinates are proprietary and the authors are not obligated to their data public There used to be a lot of months or years of involved work for each solved structure, but new streamlined methods allow for faster determination in as little as one week! (High throughput structure determination, NIH, Protein Structure Initiative.) (for more information on high throughput (not covered in class) see http://www.uwstructuralgenomics.org/) Crystals are placed into an x-ray beam. The atoms of the proteins within the crystals diffract the incident x-ray and create diffraction patterns on a film. With complex mathematical calculations crystallographers obtain an electron density map into which the amino acid sequence is fitted with help of computer graphics. L(+) lactate dehydrogenase crystals. Bar=100 µm. Ostendorp et al.(1996) Protein Science 5, 862 pentalenene synthase crystals. Lesburg et al. (1995) Protein Science 4, 2436 phosphoribulokinase crystals. Roberts et al. (1995) Protein Science 4, 2442 Diffraction Amplitude waves of diffracted electrons can add or subtract to each other. The result are the white dots on the diffraction image. Diffraction image from a pentalenene synthase crystal. Lesburg et al. (1995) Protein Science 4, 2436 Electron density mapBiochem 660 - 2008 Molecular)Graphics)Essentials)17 3 The)Protein)Data)Bank)(PDB) ) : ) web)repo sitory)of)published)st ructures ) 3.1 The web site "The Protein Data Bank (PDB) is an archive of experimentally determined three-dimensional structures of biological macromolecules, serving a global community of researchers, educators, and students. The archives contain atomic coordinates, bibliographic citations, primary and secondary structure information, as well as crystallographic structure factors and NMR experimental data." On October 14, 2008 there were 53660 PDB entries. While the majority of published structures are derived from X-ray crystallography. However, every year a larger number are derived from NMRexperiments. The number of structure has been increasing exponentially since the first structures deposited in 1972. The PDB database home page is at http://www.rcsb.org/pdb The “PDB Statistics “ button on the top left of the page leads to more information about released entries. 3.2 PDB file names Sequences are found by their “accession number.” Similarly PDB files are designated by a PDB ID code, alphanumeric and only 4 characters long. Most authors publish the PDB ID within their publications. Many proteins are represented multiple times within the database, as reported by different authors or as mutants or because they were crystallized under different conditions such as pH. For example myoglobins account for 277 entries and hemoglobins 405. The citing of a PDB structure is done by referencing the PDB ID code, followed by the primary journal citation. See http://www.rcsb.org/pdb/static.do?p=general_information/about_pdb/policies_references.html18 Molecular)Graphics)Essentials) Excerpt from web site: Structures should be cited with the PDB ID and the primary reference. For example, structure 102L should be referenced as: PDB ID: 102L D.W. Heinz, W.A. Baase, F.W. Dahlquist, B.W. Matthews How Amino-Acid Insertions are Allowed in an Alpha-Helix of T4 Lysozyme. Nature 361 pp. 561 (1993) Structures without a published reference can be cited with the PDB ID, author names, and title: PDB ID: 1CI0 Shi,