S. Takeda, A. Yamashita, K. Maeda, Y. Maeda Structure of the core domain of human cardiac troponin in the Ca2+- saturated form S. Takeda, A. Yamashita, K. Maeda, Y. Maeda Nature, 424: 35-41, July 2003

Hanging vs. Sitting Drop Technique The most common setup to grow protein crystals is by the hanging drop technique : A few microliters of protein solution are mixed with an about equal amount of reservoir solution containing the precipitants. A drop of this mixture is put on a glass slide which covers the reservoir. As the protein/precipitant mixture in the drop is less concentrated than the reservoir solution (remember: we mixed the protein solution with the reservior solution about 1:1), water evaporates from the drop into the reservoir. As a result the concentration of both protein and precipitant in the drop slowly increases, and crystals may form. There is a variety of other techniques available such as sitting drops, dialysis buttons, and gel and microbatch techniques. Robots are useful for automatic screening and optimization of crystallization conditions.  We have implemented a web computing service of Brent Segelke's CRYSTOOL, an inherently efficient random screen for crystallization conditions that you can customize.  The main advantage is the small sample size a crystallization robot can handle reproducibly, but it needs some effort to set it up. Click here to learn more about the IMPAX-II robot. Sitting Drop Images from TriTek Corp.

Crystals Native – hanging drop vapour diffustion method (CaCl2 in reservoir solution) Strontium-substituted – hanging drop vapour diffusion method (SrCl2 in reservoir solution) Osmium-derived – soaked native crystal with OsCl3 in reservoir, then flash froze

Troponin Three subunits Three samples Troponin T – binds tropomyosin Troponin I – inhibitory subunit Troponin C – binds Ca2+ Three samples Tn46KA Tn46KB Tn52KA Tn52KB

MAD aka Multiwavelength Anomalous Diffraction. Change x-ray energy (wavelength). Changes anomalous scattering factor values. Which changes the magnitudes. Needs three different measurements. The theory of MAD was developed as early as that of MIR. In MAD changes are induced in the atomic scatering factor of a heavy-atom bound to protein by measuring diffraction data at a number of different X-ray energies where the anomalous scattering factors of the heavy atom are significantly different from one another. The largest changes in the anomalous scattering factors of atoms occur around characteristic absorption edges since their values are directly related to the atoms atomic absorption coefficient. This is convenient since the majority of heavy atoms which bind to proteins have absorption edges which lie in the energy range typically used for protein diffraction work (5-20 keV). Information about the phase of the scattered X-rays can be derived from the resonance effects or anomalous scattering. Anomalous scattering is an atomic property and thus thus enters the equations for X-rays diffraction in the expression for the atomic scattering factor (f), which is the sum of "normal" atomic scattering factor f(0) and a complex anomalous cortrrection having real (f') and imaginary (f") components: f = f(0) + f' + f" Wavelength-tunable synchrotron radiation allows the real component (f') to be used as well, providing the opportunity for direct phasing through combination of orthogonal effects of f' and f". The formulation of the MAD observational equation is based on that of Karle [7] and modified by Hendrickson [8]:

Diffraction Data Collection and Structural Analysis Part I Diffraction patterns were collected. Initial phases where obtained at 3.3A by MAD using osmium derivative data set. Heavy atom positions were searched for by SOLVE. Phases were calculated by SHARP, then improved by SOLMON. Model building was done using TURBO-FRODO. Could trace almost all of the chains, but quality was not good enough to do detailed model building.

Diffraction Data Collection and Structural Analysis Part II Used another set of MAD data from strontium (Sr2+)-substituted crystals at 2.8A, which was enough for model building. Sr2+ ions were in the Ca2+ binding sites (II-IV). Structural models were used for model building. Structure was refined against native Tn46K data set at 2.6A resolution. Several rounds of model building and refinement. Final structural model for two Tn46K molecules.

Diffraction Data Collection and Structural Analysis Part III Used molecular replacement (using refined Tn46K model) to solve Tn52K. Several rounds of model building and refinement. Final structural model for two Tn56K molecules.

Refinement Statistics