June 10, 2003

Bio-matters: from IR to X-rays. (including information about the Crystallization Workshop)

For the past two decades the National Synchrotron Light Source (NSLS) has been increasingly contributing to structural biology. With the advent of a new facility the aim of this workshop was to discuss the contributions of different synchrotron radiation-based methods to the understanding of the molecular structure and bio-molecule function. The second goal was to focus on the complementary aspects between these techniques and different methods such as cryo-electron microscopy and neutron scattering methods. The workshop consisted of oral presentations, a poster session and a panel discussion session on the future requirements and expectations of the NSLS user community. The talks presented are summarized below:

Wayne Hendrickson, Columbia University, “Synchrotron Crystallography in Biological Discovery”, introduced the subject of the workshop. In his talk he described the impact of synchrotron radiation on the field of biological crystallography, a number of technical advances and the problems of radiation damage with the advent of more intense sources. Several examples were discussed in relation to the speed of solution provided by crystallography at synchrotron radiation sources, and the impact to biochemistry and molecular biology.

Chris Jacobsen, Stony Brook University, “Soft x-ray imaging and spectromicroscopy”, presented high resolution views of chemical contrast through the combination of soft x-ray microscopes and near-edge spectroscopy methods. This approach was illustrated with biomedical examples including microspectroscopy studies of human sperm, and imaging of several cell types.

Rob Scarrow, Haverford College,“EXAFS studies of metalloproteins and the usefulness of model coordination complexes”, discussed the application of EXAFS (Extended X-ray Absorption Fine Structure) analysis to a variety of metalloproteins. The determination of the nature of ligand atoms, the number and lengths of bonds, and metal-metal distances and how small molecule crystal structure databases are useful in the interpretation of the results was discussed using lipogenase and porphobilinogen synthase as examples.

Joannna Krueger, University of North Carolina at Charlotte, ”Small-angle scattering: solutions in protein structural analysis”, discussed X-ray and neutron small angle scattering focusing on the complementary aspects of these techniques and other structural and biochemical approaches such as that obtained from selected-site mutagenesis, circular dichroism, NMR, and electron microscopy.

Udupi A. Ramagopal, Albert Einstein College of Medicine, and Zbigniew Dauter, NIH, “SAD: Happy phasing with weak anomalous scatterers”, described the single-wavelength anomalous diffraction (SAD) as an alternative to the multiple wavelength diffraction method (MAD) applied to sulfur containing proteins and to radiation sensitive samples.

Uwe Bergmann, Stanford University, “Advances in High-Resolution Hard X-ray Spectroscopy: From Vibrational Studies to identify ligands to the Local Structure of Water”, explained that hard X-ray spectroscopy became possible in recent years due to intense sources and improvements in X-ray instrumentation. The application of X-ray fluorescence spectroscopy (XFS) of weak lines, resonant inelastic X-ray scattering (RIXS), (non resonant) X-ray Raman scattering (XRS) and nuclear resonant vibrational spectroscopy (NRVS) to studies of the oxygen K-edge of water, metalloproteins and Fe containing systems was shown.

Mark Chance, Albert Einstein College of Medicine, “Structure and Dynamics of Macromolecular Machines”, described synchrotron foot-printing to study the dynamics and interactions of proteins and nucleic acid structures with millisecond time resolution and high structural resolution using nanomoles to picomoles of material. He gave examples for the L-21 ribozyme from Tetrahymena, cofilin and time-resolved activation of the actin binding protein gelsolin.

Lisa Miller, National Synchrotron Light Source, “ Chemical imaging of biological tissues using a combination of infrared, UV-visible fluorescence, and x-ray micro-spectroscopy”, discussed the application of synchrotron infrared (IR) micro-spectroscopy and fluorescence techniques for examining the inherent chemical makeup of biological cells and tissues at spatial resolutions not achieved by conventional IR microscopes. Comparisons with other techniques such as immunofluorescence and X-ray micro-spectroscopy were presented in light of Alzheimer’s disease, scrapie, and bone disease.

Thomas C. Terwilliger, Los Alamos National Laboratory, ”Structural Genomics: Technology for Structural Biology”, presented the future needs of structural genomics and the current status. He focused on the technological improvements needed from protein production to structure determination. Several of these developments are underway, one of the most important being the automation of data collection and analysis at X-ray beam lines worldwide. Other technologies such as the engineering of proteins for optimal solubility, automated structure solution and phase improvement by X-ray crystallography were also discussed.

Several posters on different subjects ranging from imaging to scattering and from the NASA radiological program to Cryo Electron Microscopy were discussed over coffee and lunch break.

Members of the panel (Wayne Hendrickson, Columbia University; Thomas Terwilliger, Los Alamos National Laboratory; Joachim Frank, Wadsworth Center; and Naomi Chayen, Imperial College, UK) and workshop participants addressed several technological problems such as instruments, detectors, methods and software developments to subjects such as multiple assemblies and unstable systems. The main recommendations were related to the development of detectors, brighter sources, instrumentation to handle smaller crystals, software for automated structure determination, modeling and docking.

Prior to the Bio-matters workshop, a workshop was held on the basic and advanced methods in protein crystallization. The aim of this one-day workshop was to allow participants to have a hands-on experience with the different crystal growth methods available to protein crystallographers. Naomi Chayen ( Imperial College, UK) explained the microbatch method and the oil method; Miroslawa Dauter (NIH) discussed the hanging drop method, co-crystallization of heavy atoms and seeding; Zbigniew Dauter (NCI-NIH) presented strategies in choosing an optimal derivative and data collection; and Grahemen Williams (Brookhaven Instruments) discussed the application of the light scattering technique to protein crystallization. Two parallel sessions were organized in the morning and in the afternoon where the 22 participants could experience the different crystallization methods. We thank our sponsors Nextal Biotechnologies, Brinkmann Instruments, Millipore, Fisher Scientific, Brookhaven Instruments Corporation, and New York New Jersey Scientific, Inc., for their kind support, without which the Crystallization Workshop, a satellite meeting to the NSLS Annual Users’ Meeting would not have been made possible.

FOR MORE INFORMATION
Lead Author:
Vivian Stojanoff
Building 725D
NSLS
Brookhaven National Laboratory
Upton, NY 11973-5000
Tel.: (631) 344-8375
Fax: (631) 344-3238
Email: stojanoff@bnl.gov

Wolfgang Caliebe
Building 725D
NSLS
Brookhaven National Laboratory
Upton, NY 11973-5000
Tel.: (631) 344-4744
Fax: (631) 344-3238
Email: caliebe@bnl.gov