Phasing HomeLab™
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View a slide presentation to learn how Cr phasing can contribute to the success of your protein structure determination program. |
Anomalous scattering with soft X-ray radiation creates new possibilities in phasing for macromolecular crystallography. The heart of the Phasing HomeLab is the FR-E DW SuperBright™ generator, the only microfocus rotating anode laboratory X-ray source featuring controlled wavelength selection. The FR-E DW SuperBright provides a cost-effective path to enhanced homelab single-wavelength anomalous diffraction (SAD) phasing while retaining traditional data collection capabilities.
Other Rigaku rotating anode generators can also be equipped with Chromium (Cr) targets to provide the foundation for a Phasing HomeLab. All Phasing HomeLabs use the same optics and choice of detector. VariMax™ Cr optics maximize the beam produced by the Rigaku generators and deliver it exactly where it is needed: on the crystal. Incident and exit helium beam paths reduce air absorption and reduce background scatter. Since Cr radiation expands the diffraction pattern relative to Cu, a large aperture detector, such as the Rigaku R-AXIS IV++ or R-AXIS HTC. The X-stream™ 2000 low temperature system maintains the crystals at cryogenic temperatures without the need for a house supply of liquid nitrogen.
Since Cr radiation expands the diffraction pattern relative to Cu, a large aperture detector, such as the Rigaku R-AXIS IV++ or R-AXIS HTC is recommended.
| Source: | FR-E
DW SuperBright microfocus rotating anode generator VariMax optics |
| Detector: | R-AXIS IV++ two-IP system R-AXIS HTC three-IP system |
| Goniometer: | Single φ vertical or 3-axis partial χ goniometer for R-AXIS detectors |
| Software: | CrystalClear™
for data collection and processing StructureStudio™ for data collection d*TREK® and HKL-2000® for data processing |
| Low temp: | X-stream 2000 for worry-free sample cooling without liquid nitrogen |
Just released: The crystal ctructure of cytochrome P460 of Nitrosomonas europaea reveals a novel cytochrome fold and heme-protein cross-link
Who should use Chromium radiation?
Investigators who wish to increase the throughput of de novo structure solution in their home laboratory. This would include investigators involved in the structural genomics initiative who wish to solve structures between synchrotron trips.
Chromium radiation (2.29 Å) doubles the available anomalous signal from elements such as S, Ca and Se as compared to the signal available with copper radiation (1.54 Å). This enhanced signal has allowed the structures of thaumatin and trypsin to solved using that signal with relatively small data sets, 45° and 180°, respectively. Work is currently progressing on the structure of glucose isomerase using only Cr radiation enhanced data.
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A survey of the PDB shows the number de novo structures solved by MAD has been decreasing steadily since 2000. However, the number of SAD has been steadily increasing. From the trends shown in the plot above we expect that more structures will be solved by SAD than MAD in 2005. Personal communication, B.C. Wang. |
The chromium radiation enhanced anomalous signal may also be used to augment SIRAS data that is insufficient to phase the data alone. In effect, using chromium radiation adds a second heavy atom, which concomitantly reduces the phase error and in turn yields more easily interpretable electron density maps. This procedure was essential to phase the structures of two proprietary proteins in our home lab.
Table 1. Increase in ƒ" for sulfur, calcium and selenium with chromium radiation versus copper radiation. |
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Table 2. Experimental improvement in <ΔF>/<F> thaumatin, trypsin and glucose isomerase for chromium radiation versus copper radiation. | |||||||||||||||||||||||||||
How do you use Chromium radiation?
The best way to start using chromium radiation in your home lab is upgrade your current facility with the components in our Phasing HomeLab.
Note: Cr anodes are available for all Rigaku generators, so at-home phasing is not restricted to the FR-E DW SuperBright.