Research Interests

Crystallization of biological macromolecules

Understanding and Developing Methods for the Crystallization of Biological Macromolecules

Determining the three dimensional structure of a protein molecule is a first step in understanding how that molecule functions.  One method used to determine the structure of protein molecules is X-ray crystallography.  X-ray crystallography is a linear process.  Progress will stop if you fail to complete any of the necessary steps that eventually lead to the molecular structure.  Protein crystallization is one of those steps and is often regarded as a bottleneck in structure determination.

Crystallization depends on many variables.  To crystallize a protein you must drive a protein solution toward a state of supersaturation.  Under the correct solution conditions the protein will form crystals.  These crystals will act as a three-dimensional X-ray diffraction grating and allow scientists to determine the molecular structure.  The detailed representations of the macromolecules allow researchers to understand how the molecule functions.

Through an active and nearly two-decade long collaboration with Dr. George T. DeTitta a basic understanding of certain physical principles behind macromolecular crystallization methods have been discovered.  The knowledge gained from this research helped to understand subtle variations in what were originally thought to be identical experiments that produced very different outcomes.  This improved understanding enabled us to develop new crystallization methods and helped other researchers to better understand and reproduce crystals in their own laboratories.

Unfortunately, we do not yet have a rational approach that allows an investigator to take a novel protein and predict its crystallization parameters.  Instead we rely on intensive screening of chemical and physical conditions for protein crystallization.  Investigators will often set up hundreds of experiments in the hope that one or more of them will lead to the formation of crystals.  Each of these experiments is unique, varying the conditions that affect protein solubility.  The outcome of the majority of these experiments will not be crystalline.  However, if the outcome of even one of these experiments provides a useful lead for producing the high quality crystals required for structure determination, the screening is considered a success.

A high throughput laboratory designed to search for initial crystallization was developed at HWI through collaboration with Dr. George T. DeTitta. Using this method we are able to set up every protein with 1536 unique crystallization conditions in less than ten minutes time.  This has proven to be an effective strategy to determine initial crystallization conditions.  This laboratory has enabled HWI staff to set up more than ten million crystallization experiments on 6500 samples.  Both the structural biology (http://www.chtsb.org/) and structural genomics (http://www.nesg.org/) (http://www.sgpp.org/) communities are being served by this research. 

We are working to bring these same high throughput methods to subsequent stages of the crystallization pipeline.  Our goal is to take samples that produced leads from the two hundred samples sent to the screening laboratory each month (largely from a group of 600 structural biologist collaborators) and produce crystals that diffract X-rays well enough to answer the biological question that is under investigation.  We are developing these methods in high throughput so as not to restrict the output of our screening laboratory.  After identifying optimized crystallization conditions in traditional 1536 well plates, we intend to produce crystals in a container that is directly suitable for X-ray diffraction experiments (http://www.chtsb.org/).

SELECTED PUBLICATIONS

Luft’s publications include total of approximately 70 papers & chapters, 140 abstracts.

• Luft, J.R. and DeTitta, G.T., HANGMAN: A Macromolecular Hanging Drop Vapor Diffusion Technique, Lab Notes, J. Appl. Cryst., 25, 324-325 (1992).
• Luft, J.R. and DeTitta, G.T., Kinetic Aspects of Macromolecular Crystallization, Methods of Enzymology, 276, 110-131 (1997).
• Luft, J.R. and DeTitta, G.T., A Method to Produce Microseed Stock for Use in the Crystallization of Biological Macromolecules, Acta Cryst., D55, 988-993 (1999)
• Luft, J.R., Rak, D.M., and DeTitta, G.T., Microbatch Macromolecular Crystallization on a Thermal Gradient, J. Crystal Growth, 196, 447-449 (1999).
• Luft J.R., Wolfley, J.,. Jusrisica I., Glasgow J., Fortier, S., and DeTitta G.T., Macromolecular Crystallization in a high throughput laboratory-the search phase, J. Crystal Growth, 232, 591-595 (2001)
  
• Luft, J.R., Collins, R.J., Fehrman, N.A., Lauricella, A.M., Veatch, C.K., and DeTitta, G.T., A deliberate approach to screening for initial crystallization conditions of biological macromolecules, Journal of Structural Biology, 142, 170-179 (2003).