We have collaborated with more than 1,000 investigators working on biologically, and often medically important problems. These investigators send HWI samples to identify initial crystallization conditions. The crystallization laboratory has ‘launched many ships’ by making this first step towards structure determination. When scientists can see these biological machines, their structures, they can better understand how they function. Crystallization may seem like basic research but it is actually a stepping stone that leads to breakthroughs and improvements in the scientific understanding of disease processes which leads to worldwide advancements in health and well-being. Our research provides a foundation that allows scientists “to acquire new knowledge to help prevent, detect, diagnose, and treat disease and disability”.
The Hauptman-Woodward Institute developed one of the world’s first high-throughput crystallization laboratories where small quantities of biological macromolecules are rapidly combined with 1,536 chemical reagents and periodically imaged to identify initial crystallization conditions. This is an important first step towards producing crystals that can be used for structural investigations by X-ray crystallography. To date more than 16,000 biological macromolecules have been sent to our laboratory by investigators from around the world. We have been in continuous operation and available to the scientific community since 2000. During that time we have significantly improved our reagents, crystal detection methods, and tools for analyzing the crystallization data. We have a very good chemically diverse set of reagents to identify initial crystallization conditions for even the most challenging samples. When we produce crystals, even those that are too small to see with a microscope, we can verify their presence using femtosecond, pulsed-laser technology on a modified, state-of-the-art instrument. Our group’s research focuses on the crystallization of biological macromolecules. X-ray crystallography is the predominant method used to visualize the machinery of life, biological macromolecules, whose interactions and operations determine the health of an individual. The production of high-quality crystals is a required sample preparation step, and is often an impediment to this visualization. Our research develops methods to reliably prepare high-quality crystals for biologically and medically important targets.
1) Crystallographic Structural Investigations: My research efforts have focused on the crystallization and characterization of biologically important macromolecules for structural investigation by X-ray crystallography. During the 1980’s and 1990’s these efforts involved purification, biophysical characterization, crystallization screening, crystal optimization and X-ray diffraction studies of high-interest, biomedically relevant proteins. These included transthyretin (TTR) from a variety of species to structurally investigate inhibitor binding, the important cancer-chemotherapeutic target, human dihydrofolate reductase (DHFR) and AIDS opportunistic infection target Pneumocystis carinii DHFR andBacillus thuringiensis δ-endotoxins including Cry3Bb1, whose structure was used to study its insecticidal effects for agricultural research. From 2000 onward, the majority of my efforts have been driven by developing methods for the identification of initial crystallization conditions for structural biologists and structural genomics centers. These initial crystallization conditions provide a critical early step towards structure determination that led to more than 1,000 PDB depositions. Cody V, Luft JR, Ciszak E, Kalman TI, Freisheim JH. Crystal-Structure Determination at 2.3-Angstrom of Recombinant Human Dihydrofolate-Reductase Ternary Complex with NADPH and Methotrexate-Gamma-Tetrazole. Anti-Cancer Drug Des. 1992;7(6):483-91. http://www.ncbi.nlm.nih.gov/pubmed/1285840 Cody V, Galitsky N, Rak D, Luft JR, Pangborn W, Queener SF. Ligand-induced conformational changes in the crystal structures ofPneumocystis carinii dihydrofolate reductase complexes with folate and NADP(+). Biochemistry-Us. 1999;38(14):4303-12. http://www.ncbi.nlm.nih.gov/pubmed/10194348 Wojtczak A, Cody V, Luft JR, Pangborn W. Structures of human transthyretin complexed with thyroxine at 2.0 angstrom resolution and 3',5'-dinitro-N-acetyl-L-thyronine at 2.2 angstrom resolution. Acta Crystallogr D. 1996;52:758-65. http://www.ncbi.nlm.nih.gov/pubmed/15299640 Wojtczak A, Cody V, Luft JR, Pangborn W. Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 angstrom resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr D. 2001;57:1061-70. http://www.ncbi.nlm.nih.gov/pubmed/11468389 Galitsky N, Cody V, Wojtczak A, Ghosh D, Luft JR, Pangborn W, English L. Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis. Acta Crystallogr D. 2001;57:1101-9. http://www.ncbi.nlm.nih.gov/pubmed/11468393 Larson ET, Deng W, Krumm BE, Napuli A, Mueller N, Van Voorhis WC, Buckner FS, Fan E, Lauricella A, DeTitta G, Luft J, Zucker F, Hol WGJ, Verlinde CLMJ, Merritt EA. Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity. J Mol Biol. 2008;381(4):975-88. http://www.ncbi.nlm.nih.gov/pubmed/18602399 Grant TD, Luft JR, Wolfley JR, Snell ME, Tsuruta H, Corretore S, Quartley E, Phizicky EM, Grayhack EJ, Snell EH. The Structure of Yeast Glutaminyl-tRNA Synthetase and Modeling of Its Interaction with tRNA. J Mol Biol. 2013;425(14):2480-93. http://www.ncbi.nlm.nih.gov/pubmed/23583912 2) Chemical and Physical Investigations of Equilibration: During the 1990’s, while continuing to crystallize biological macromolecules, my research efforts focused on the characterization of equilibration rates for vapor-diffusion experiments. Vapor-diffusion crystallization methods are widely used to produce crystals for structural studies. This method relies on the dehydration of a small experiment drop by a large reservoir solution to drive the biological macromolecule to a sufficiently supersaturated state for crystallization. Our aim was to determine the rate-limiting step in vapor equilibration, which turned out to be transport of water through the vapor space. Many of these studies were isopiestic measurements, typically taking many months to complete using variables such as pressure, temperature, chemical species, chemical concentration and the distance from an experiment drop to a reservoir solution. This fundamental work was largely supported by NASA and provided isopiestic data that is especially relevant for crystallization. We used this knowledge to develop passive experimental methods to adjust the rates of equilibration, an important variable that directly impacts the rate of supersaturation and can be exploited to optimize crystal volume and diffraction quality for both vapor and liquid-diffusion based crystallization methods. Arakali SV, Easley S, Luft JR, Detitta GT. Time Courses of Equilibration for Ammonium-Sulfate, Sodium-Chloride and Magnesium-Sulfate Heptahydrate in the Z/3 Crystallization Plate. Acta Crystallogr D. 1994;50:472-8. doi: Doi 10.1107/S0907444994001770. PubMed PMID: WOS:A1994NY47500025. http://www.ncbi.nlm.nih.gov/pubmed/15299405 Arakali SV, Luft JR, Detitta GT. Non-ideality of Aqueous-Solutions of Polyethylene-Glycol - Consequences for Its Use as a Macromolecular Crystallizing Agent in Vapor-Diffusion Experiments. Acta Crystallogr D. 1995;51:772-9. http://www.ncbi.nlm.nih.gov/pubmed/15299808 Detitta GT, Luft JR. Rate of Water Equilibration in Vapor-Diffusion Crystallization - Dependence on the Residual Pressure of Air in the Vapor Space. Acta Crystallogr D. 1995;51:786-91. http://www.ncbi.nlm.nih.gov/pubmed/15299810 Luft JR, Detitta GT. Chaperone Salts, Polyethylene-Glycol and Rates of Equilibration in Vapor-Diffusion Crystallization. Acta Crystallogr D. 1995;51:780-5. http://www.ncbi.nlm.nih.gov/pubmed/15299809 Luft JR, Albright DT, Baird JK, DeTitta GT. The rate of water equilibration in vapor-diffusion crystallizations: Dependence on the distance from the droplet to the reservoir. Acta Crystallogr D. 1996;52:1098-106. http://www.ncbi.nlm.nih.gov/pubmed/15299569 3) Crystallization Method Development - Gadgets: Method development for the crystallization of biological macromolecules has been an underlying theme of my research for the past 30 years. Many of these methods were developed out of necessity to solve a particular crystallization problem that related to a specific biological macromolecule, but had general applicability. Methods based on the passive control of equilibration rates that drove a protein to a state of supersaturation were largely based upon the isopiestic work described above. The method development typically used supplies available in most laboratories to increase acceptance by the wider community. Seeding methods, the introduction of nucleating agents to initiate crystal growth, were developed and included a method to pulverize large crystal for seeds. This “Seed Bead” method is still popular; after publication the device was made commercially available by Hampton Research. Luft JR, Detitta GT. Hangman - a Macromolecular Hanging-Drop Vapor-Diffusion Technique. J Appl Crystallogr. 1992;25:324-5. http://scripts.iucr.org/cgi-bin/paper?S0021889891011354 Luft JR, Arakali SV, Kirisits MJ, Kalenik J, Wawrzak I, Cody V, Pangborn WA, Detitta GT. A Macromolecular Crystallization Procedure Employing Diffusion Cells of Varying Depths as Reservoirs to Tailor the Time-Course of Equilibration in Hanging-Drop and Sitting-Drop Vapor-Diffusion and Microdialysis Experiments. J Appl Crystallogr. 1994;27:443-52. http://scripts.iucr.org/cgi-bin/paper?S0021889893012713 Luft JR, DeTitta GT. Kinetic aspects of macromolecular crystallization. Method Enzymol. 1997;276:110-31. http://www.ncbi.nlm.nih.gov/pubmed/9148108 Luft JR, DeTitta GT. A method to produce microseed stock for use in the crystallization of biological macromolecules. Acta Crystallogr D. 1999;55:988-93. http://www.sciencedirect.com/science/article/pii/S0022024898008641 Luft JR, Rak DM, DeTitta GT. Microbatch macromolecular crystallization in micropipettes. J Cryst Growth. 1999;196(2-4):450-5. http://www.sciencedirect.com/science/article/pii/S0022024898008641 Luft JR, Rak DM, DeTitta GT. Microbatch macromolecular crystallization on a thermal gradient. J Cryst Growth. 1999;196(2-4):447-9. http://www.sciencedirect.com/science/article/pii/S002202489800863X 4) High-Throughput Crystallization: My interest in method development for high-throughput crystallization took place prior to the Protein Structure Initiative; the methods were originally developed to compare a precipitation reaction index of proteins set up against a set of chemical cocktails as a means to determine protein similarity and predict crystallization behavior. Initial batch experiments, where the protein was combined with a chemical cocktail, took place in glass capillaries. In collaboration with George DeTitta, a method to rapidly set up microbatch-under-oil experiments was developed using commercially available, syringe-based, automated liquid-handling systems. A high-throughput crystallization screening laboratory was developed around this technology to set up a 1536 chemical cocktail assay, image outcomes for a period of time and to archive, distribute, view and analyze the image data. The infrastructure has been significantly improved over the years, including more effective cocktails for soluble and membrane proteins, better imaging (now able to detect submicron-sized crystals) and tools for data analysis. The laboratory, a community resource, has to date assayed >15,000 biological macromolecules for crystallization for more than 1,000 investigators. To learn more about sending a sample for crystallization screening please visit: http://getacrystal.org Luft JR, Wolfley J, Jurisica I, Glasgow J, Fortier S, DeTitta GT. Macromolecular crystallization in a high throughput laboratory-the search phase. J Cryst Growth. 2001;232(1-4):591-5. http://www.sciencedirect.com/science/article/pii/S0022024801012064 Luft JR, Collins RJ, Fehrman NA, Lauricella AM, Veatch CK, DeTitta GT. A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J Struct Biol. 2003;142(1):170-9. http://www.ncbi.nlm.nih.gov/pubmed/12718929 Luft JR, Wolfley JR, Said MI, Nagel RM, Lauricella AM, Smith JL, Thayer MH, Veatch CK, Snell EH, Malkowski MG, Detitta GT. Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature. Protein Sci. 2007;16(4):715-22. http://www.ncbi.nlm.nih.gov/pubmed/17327388 Luft JR, Snell EH, DeTitta GT. Lessons from high-throughput protein crystallization screening: 10 years of practical experience. Expert Opin Drug Dis. 2011;6(5):465-80. http://www.ncbi.nlm.nih.gov/pubmed/22646073 Luft JR, Wolfley JR, Snell EH. What's in a Drop? Correlating Observations and Outcomes to Guide Macromolecular Crystallization Experiments. Cryst Growth Des. 2011;11(3):651-63. http://www.ncbi.nlm.nih.gov/pubmed/21643490 Luft JR, Newman J, Snell EH. Crystallization screening: the influence of history on current practice. Acta Crystallogr F. 2014;70:835-53. http://www.ncbi.nlm.nih.gov/pubmed/25005076 Luft JR, Wolfley JR, Franks EC, Lauricella AM, Gualtieri EJ, Snell EH, Xiao R, Everett JK, Montelione GT. The detection and subsequent volume optimization of biological nanocrystals. Struct Dyn. 2015;2(4):041710. http://scitation.aip.org/content/aca/journal/sdy/2/4/10.1063/1.4921199 5) SAXS: Method development for high-throughput Small Angle X-ray Scattering (SAXS) took place through a collaboration with Hiro Tsuruta (Stanford), Edward Snell and Thomas Grant. The high-throughput screening laboratory had a library of structural genomics targets; leftover samples from crystallization screening, a significant number remained structurally uncharacterized by NMR or X-ray crystallography. In collaboration with the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 4-2, we worked with beamline scientists to rapidly collect SAXS data from these samples. My primary role for this project was to develop protocols for sample-handling, dilution and preparation for data collection, with a minor early role in data collection and analysis. This work enabled rapid characterization of hundreds of biological macromolecules using SAXS and showed that the majority of the samples that did not crystallize were well-folded and would have been expected to respond favorably to crystallization. We have publications to demonstrate the value of complementary SAXS data for NMR and crystallographic investigations. Grant TD, Luft JR, Wolfley JR, Tsuruta H, Martel A, Montelione GT, Snell EH. Small Angle X-ray Scattering as a Complementary Tool for High-Throughput Structural Studies. Biopolymers. 2011;95(8):517-30. http://www.ncbi.nlm.nih.gov/pubmed/21462184 Grant TD, Luft JR, Wolfley JR, Snell ME, Tsuruta H, Corretore S, Quartley E, Phizicky EM, Grayhack EJ, Snell EH. The Structure of Yeast Glutaminyl-tRNA Synthetase and Modeling of Its Interaction with tRNA. J Mol Biol. 2013;425(14):2480-93. http://www.ncbi.nlm.nih.gov/pubmed/23583912 Grant TD, Luft JR, Carter LG, Matsui T, Weiss TM, Martel A, Snell EH. The accurate assessment of small-angle X-ray scattering data. Acta Crystallogr D. 2015;70:45-56. http://www.ncbi.nlm.nih.gov/pubmed/25615859
Joseph R. Luft
T: 716 898 8623
T: 716 898 8623