Workshop on Emerging Areas in Biological Crystallography
July 27-28, 2004, APS, Argonne, Illinois
Scope
Performing protein structure determination is an endeavor geared to decipher the function of proteins in the human body. It has been argued that because the function of a protein is encoded by its three-dimensional structure, the structures will lead to the knowledge of protein function. Developments in synchrotron radiation sources over the past 20 years have revolutionized protein structure determination. The intense tunable undulator x-ray sources available at the third generation synchrotron facilities have enhanced the Multi-wavelength Anomalous Dispersion (MAD) technique for solving the phasing problem to obtain 3-D structures. This has enabled the collection of MAD data sets from favorable crystals in a few minutes. Much emphasis in recent years has been given to the effective use of synchrotron sources for protein structure studies. It consists of protein crystallization, storage, monitoring of crystal growth, harvesting and freezing crystals, robotic capabilities to mount and dismount the cryo-cooled crystals on a goniometer, improved 2-D detector systems and data acquisition. In addition, the interpretation of the x-ray diffraction data, and graphics display to enhance the quality of the molecular model have been given very high priority by the research community.
The present workshop went beyond these issues and addressed future challenges of this field of research in the following areas.
- Micro-focused x-ray beam experiments
- Structure and dynamics of macromolecular assemblies
- Time Resolved Protein Crystallography
- Powder Diffraction of Protein Complexes
- Diffuse Scattering and Nuclear Inelastic Scattering
Progress in the field of protein crystallography is often hindered by the size, shape and the quality of the available crystal. For example, crystals which are only a few tens of microns in dimension, or with a growth habit of a thin needle or a platelet, or highly mosaic or twinned will not produce quality diffraction patterns. It has been demonstrated that each of these difficulties can be overcome by the use of microfocused x-ray beam of the type routinely used in the materials research at all the third generation synchrotron radiation facilities.
Micro-focused x-ray beams also offer the possibility to study partially ordered systems such as the membrane protein arrays, such as a class of membrane-bound proteins made up of microarrays of G-protein-coupled receptors (GPCRs). These have the potential use in medical diagnostics, biomarker discovery, and proteomics. Another example for the micro-focused x-ray beam investigation is the liposomes, made up of natural nontoxic phospholipids and cholesterol. They are used as drug carriers to cure inflammation to cancer. The challenge will be to identify such systems and to address the potential and the limitations of the technique in their structure determination.
Macromolecular assemblies/molecular machine of multiple components are generally large and the structure is determined by combining electron cryomicroscopy with x-ray crystallography. The best example is the work on a clathrin coated vesicle with a resolution of about 9Å. Can this resolution be improved with the use of microfocused x-ray beam? The workshop included experts to address various aspects on this question.
Radiation Damage
A long standing issue in performing synchrotron radiation experiments with soft matter is the radiation damage of exposed samples. Over the course of years many experimental approaches to overcome this limitation have helped to make considerable progress in protein crystallography, the most popular solution being cryo-cooling of the sample. The subject has received even more attention with the expectation of performing single molecule imaging with ultrashort x-ray pulses from future x-ray FELs. It is fair to say that our limited ability to predict the behavior of protein crystals when exposed to intense synchrotron radiation beams deserves further attention. The workshop addressed this subject by defining both the experimental and theoretical research programs essential to make further advances.
The experimental approach to time-resolved protein crystallography experiments depends mainly on the kinetics of the reaction and the conformational changes exhibited by the molecule in response to external stimuli. Many successful experiments of this type have been performed at the third generation synchrotron radiation sources with nanosecond resolution. For example, the changes in biologically important effectors have been measured with nanosecond resolution in many photoreactive "caged" compounds. The workshop talks addressed both experimental and theoretical issues in relating the intermediate structural changes to the reaction steps.
High resolution powder diffraction method to determine the protein structure is a newly evolving technique. There are many advantages of this technique over single crystal diffraction work. For example, the protein powder can be formed over a wide range of conditions and time scales quite unlike the restricted circumstances required for producing large, single crystals. The powders are suitable for the study of protein-ligand complexes because of the immunity to crystal fracture and to any phase change that may accompany complex formation.
This diffuse scattering has been used to study the nature of disorder in protein crystals and has been shown to be a useful experimental technique for characterizing the fluctuations of crystalline proteins. It provides information regarding the directions of motions as well as the lengths and directions of correlations in atomic displacements. Diffuse scattering is usually thrown away in most crystallographic studies, but the availability of synchrotron sources, advanced detectors, and sophisticated computing have made it easier to use it fruitfully. Equally important method that provides dynamical information of specific atoms in a protein uses inelastic nuclear scattering method. It provides local phonon density of states from the nuclear resonance from sites occupied by labeled nuclei.
