Biology: Customized Macromolecular Crystallography
Although the main research areas of interest at BioCARS are time-resolved macromolecular crystallography and structural studies of biohazards at the BSL-2 and BSL-3 level, BioCARS also offers the full range of standard macromolecular crystallography experiments such as: single wavelength, SAD and MAD, ultra-high resolution and large unit cell data collection. The 14-BM-C station, with a large Quantum-315 ADSC detector is particularly suitable and has been very successful in ultra-high resolution and large unit cell data collection.
Our flexible end-station setup and auxiliary equipment available to users permit non-standard experiments, for example those involving on-line micro-spectrophotometry, flow cell use and on-line illumination of samples by visible light from laser and other light sources. These tools are particularly important for kinetic crystallography (Bourgeois and Royant, 2005; Petsko and Ringe, 2000; Schlichting, 2000; Stoddard, 2001), where transient, intermediate states in the course of a reaction are examined by trapping them by various physical or chemical means. This technique is closely related and complementary to ultra-fast time-resolved crystallography where short-lived intermediate states are also examined but in real time, with no trapping involved (Moffat, 2001; Schmidt et al., 2005). BioCARS laser laboratory, where off-line static and time-resolved spectroscopy on crystals can be conducted, provides important additional means of addressing questions related to kinetic crystallographic studies.
Off-line and on-line micro-spectrophotometry is proving to be an important tool for assessing the catalytic properties of enzymes in the crystal (Bourgeois et al., 2002; Pearson et al., 2007). It can be applied to metallo-enzymes, photosensitive proteins or proteins with co-factors, substrates or products that absorbs in UV-VIS. Micro-spectrophotometry can be used to confirm the presence or absence of a substrate or a co-factor, as well as the redox state of a protein. It can also aid the design of efficient triggering of a reaction in the crystals or trapping of intermediate states. On-line micro-spectrophotometry is also particularly useful for assessing radiation damage, including the unwanted redox processes triggered by intense x-ray beams, and for determining the best data collection strategy for avoiding the extensive radiation damage (Berglund et al., 2002; Pearson et al., 2007).
- Berglund, G.I., Carlsson, G.H., Smith, A.T., Szoke, H., Henriksen, A., and Hajdu, J. The catalytic pathway of horseradish peroxidase at high resolution. Nature 417, 463-468 (2002) abstract
- Bourgeois, D., and Royant, A. Advances in kinetic protein crystallography. Current Opinion in Structural Biology 15, 538-547 (2005) abstract
- Bourgeois, D., Vernede, X., Adam, V., Fioravanti, E., and Ursby, T. A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals. Journal of Applied Crystallography 35, 319-326 (2002) abstract
- Moffat, K. Time-resolved biochemical crystallography: a mechanistic perspective. Chemical Reviews 101, 1569-1581 (2001) abstract
- Pearson, A.R., Pahl, R., Kovaleva, E.G., Davidson, V.L., and Wilmot, C.M. Tracking X-ray-derived redox changes in crystals of a methylamine dehydrogenase/amicyanin complex using single-crystal UV/Vis microspectrophotometry. Journal of Synchrotron Radiation 14, 92-98 (2007) abstract
- Petsko, G.A., and Ringe, D. Observation of unstable species in enzyme-catalyzed transformations using protein crystallography. Current Opinion in Chemical Biology 4, 89-94 (2000) abstract
- Schlichting, I. Crystallographic structure determination of unstable species. Accounts of Chemical Research 33, 532-538 (2000) abstract
- Schmidt, M., Ihee, H., Pahl, R., and Srajer, V. Protein-ligand interaction probed by time-resolved crystallography. Methods in Molecular Biology (Clifton, N.J 305, 115-154 (2005) abstract
- Stoddard, B.L. Trapping reaction intermediates in macromolecular crystals for structural analyses. Methods (San Diego, Calif 24, 125-138) (2001) abstract
Colored protein crystal in a cryo-loop
The 4DX micro-spectrophotometer optics mounted on-line at the 14-BM-C beamline