Biology Reference
In-Depth Information
plastic film and incubated at a fixed temperature, usually around 20
C. The drops
are observed on a daily basis with a stereo microscope to detect any crystal growth.
Numerous crystallization kits in a 96-wellformatareprovidedbyseveralcompa-
nies. They correspond to a collection of diverse kinds of buffers, precipitants, and
additives designed to cover a wide range of conditions. The number of screens to be
used depends on protein availability. The crystals obtained at this step are generally
small (
100
m
m) or not well shaped and need to be optimized through refinement
of the preliminary crystallization conditions. This refinement is performed by gen-
erating “home-made” grid screens where all the parameters of the initial hit are
varied (pH, salt, concentration of precipitant, etc.). Typically, this optimization
step is carried out manually in 24-well crystallization plates by mixing 1
m
Lof
the concentrated protein with 1
m
L of each crystallization condition, the reservoir
containing 500
m
L of the corresponding crystallization condition. The drops are
suspended from siliconized glass coverslips, which are used to seal the wells with
Vaseline (hanging drop method). This scale-up of the crystallization process gen-
erally provides larger crystals suitable for further crystallographic studies. Once
they have reached suitable sizes, the crystal are mounted into a cryogenic nylon
loop and flash frozen in liquid nitrogen to preserve it before and during the
X-ray diffraction experiment. This is achieved by transferring the crystal to a solution
containing the mother liquor plus 20-30% cryoprotectant (e.g., glycerol) for a few sec-
onds before scooping up the crystal into the loop again and rapidly freezing by plung-
ing it into liquid nitrogen. The entire process is carried out by looking through a stereo
microscope. The frozen crystal is then stored in a dewar maintained at cryogenic
temperature until data collection. Regarding the example of the RAR
a
-RXR
a
LBD
heterodimer crystallization, the initial crystals were observed under condition
#38 of the Crystal Screen II (Hampton Research) comprising 20% PEG 10,000 and
0.1 M HEPES, pH 7.5. The crystals grew as small hexagonal bipyramids of
50
<
25
m
m
3
within 5 days. Further attempts to optimize the conditions were per-
formed by using PEGs of various molecular weights, additives at different concentra-
tions, and pH levels ranging between 6.0 and 8.5. Optimized crystallization conditions
contained 23% PEG 10,000, 0.1 M HEPES, pH 7.25, the reservoir being composed of
20% PEG 10,000, 0.1 M HEPES, pH 7.25. Under such conditions, only a few large
crystals (500
25
250
m
m
3
) were obtained. At this step, the structure is solved
by conventional crystallographic analysis with the collection of diffraction data using
in-house or synchrotron X-ray sources, followed by data processing, determination of
the structure, model building, and refinement (see
Bourguet, Vivat, et al., 2000
for
more details and programs used).
250
2.1.2
Expression and purification of NR-NR complexes
The availability of homogeneous NR dimers is a prerequisite for biochemical and
biophysical studies addressing NR-NR interactions. We and others have developed
similar methods of overexpression and purification of high-quality NR-NR
complexes (
Bourguet, Andry, et al., 2000; Iyer et al., 1999
). These protocols apply
