Phasing protein structures at home using Cr radiation
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View a slide presentation to learn how Cr phasing can contribute to the success of your protein structure determination program. |
The single-wavelength anomalous dispersion method (SAD) is frequently used to solve protein structures from synchrotron data by taking advantage of the tunable wavelength to collect heavy atom derivatives near the metal's absorption edge. For methionine-containing proteins, selenomethionine derivates may be prepared to take advantage of the anomalous Se signal.
However, some proteins will not crystallize as SeMet derivatives, and not all proteins contain methionine. Sulfur, on the other hand, is present in almost all proteins in cysteine residues. The process of structure solution is greatly simplified if SeMet substitution or heavy-atom soaks are not necessary.
The structure of crambin was solved in 1981 using the resolved atom S-SAD method and Cu Kα radiation and a number of other proteins have been solved using Cu or synchrotron radiation tuned to near-Cu wavelengths.
The ƒ" for S with Cu radiation is fairly small, 0.56 e-. Using a longer wavelength radiation, such as Cr Kα (λ=2.29 Å), enhances the anomalous signal, with an ƒ" value of 1.14 e-. However, air, loop and buffer absorption is more pronounced with Cr radiation and the diffraction pattern is spread out over a wider 2θ range, so more care must be taken when performing the experiment.
To maximize the signal-to-noise ratio for the experiment, thereby increasing the significance of the sulfur anomalous signal, a high-intensity source is required. Rigaku's FR-E+ DW SuperBright™ dual wavelength microfocus rotating anode generator, the highest-flux chromium source available, features computer-controlled wavelength selection between Cu and Cr, providing a single X-ray source that allows users to easily switch between these two radiations. VariMax™ Cr optics maximize the beam produced by the Rigaku generators.
Large aperture area detectors, such as the Rigaku R-AXIS IV++ or R-AXIS HTC, can be fitted with incident and exit helium beam paths reduce air absorption and reduce background scatter.
The best way to start using chromium radiation in your home lab is upgrade your current facility with the components in Rigaku's Phasing HomeLab™.
Note: Cr anodes are available for all Rigaku generators, so at-home phasing is not restricted to the FR-E+ DW SuperBright.
