Cr-Phasing HomeLab™
|
|
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 a Cr-Phasing HomeLab is any Rigaku rotating anode generator with a chromium anode..
All Rigaku rotating anode generators can be equipped with chromium (Cr) targets to provide the foundation for a Cr-Phasing HomeLab. All Cr-Phasing HomeLabs use the same optics and choice of large aperture 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, is recommended.
|
Source: |
Any Rigaku rotating anode generator
with Cr target |
|
Detector: |
R-AXIS IV++ two-IP system |
|
Single φ (vertical or inverted) goniometer for R-AXIS detectors |
|
|
Software: |
CrystalClear™
for data collection and processing |
Structures solved by Cr phasing at Nagoya University
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.
|
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. |
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. |
|
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 to upgrade your current facility. Current Rigaku rotating anode generator owners can easily convert to a Cr-Phasing HomeLab by adding a Cr anode and VariMax Cr optics.
Some Cr -SAD publications:
- Yu Kitago, Nobuhisa Watanabe and Isao Tanaka, Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method, Acta Cryst. (2005). D61, 1013-1021
- Acta Crystallogr D Biol Crystallogr. 2003 Nov;59(Pt 11):1943-57. Epub 2003 Oct 23. Away from the edge: SAD phasing from the sulfur anomalous signal measured in-house with chromium radiation.
- Acta Crystallogr D Biol Crystallogr. 2005 Jul;61(Pt 7):960-6. Epub 2005 Jun 24. Away from the edge II: in-house Se-SAS phasing with chromium radiation.
- Acta Crystallogr D Biol Crystallogr. 2005 Aug;61(Pt 8):1013-21. Epub 2005 Jul 20. Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method.
- Acta Crystallogr D Biol Crystallogr. 2005 Jul;61(Pt 7):960-6. Epub 2005 Jun 24. Away from the edge II: in-house Se-SAS phasing with chromium radiation.