Successful
protein crystallography typically requires that crystals be cryocooled
in order to reduce radiation damage at the data collection stage. So
far, protein crystals have most commonly been frozen by flash cryocooling
at ambient pressure, which often requires a time-consuming search for
cryoprotection conditions. Recently, the CHTSB-affiliated team (led
by Prof. Sol Gruner) at the Cornell High Energy Synchrotron Source
(CHESS) has developed an alternative procedure, high-pressure cryocooling,
which does not require the addition of chemical cryoprotectants. The
result of high-pressure cryocooling is very often a dramatic improvement
in the quality and resolution of the diffraction data as seen in Fig.
1. The Cornell team is currently investigating the basic underlying
principles and experimental parameters important for the optimization
of the high pressure cooling method.
Since the high-pressure
method involves the use of helium gas as a pressurizing medium, attention
has been focused on its extension to diffraction phasing by incorporating
heavy noble gases such as krypton or xenon. In the test case of Porcine
Pancreatic Elastase (PPE, 240 residues, 26kDa), very high quality diffraction
was obtained by the modified high-pressure cyrocooling method without
the help of cryoprotectants. Furthermore, a single krypton site with
an occupancy of 0.31 could be used successfully for SAD phasing at
1.3Å resolution
(Fig. 2). The Cornell team is currently working on equipment modifications
that will be compatible with a high throughput crystallography pipeline. Fig.
2. A section of the electron density map for PPE contoured at 1s
before density modification at 1.3Å resolution.
The final refined model of PPE was superimposed for map evaluation. After
density modification, a model-building program, ARP/wARP, could automatically
trace 220 residues out of 240. |
|
 |
Fig1. X-ray diffraction
patterns with frozen specimen at 1.9 kbar (left) with no cryoprotectants.
Diffracts to a 2.7Å mosaicity 0.6°. Flash frozen (right) using
no cryoprotectants. Diffracts to only 7Å with poor mosaicity. (Acta
Cryst. D61. 2005. 881-890) |
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Fig. 2 |
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