Biology Reference
In-Depth Information
experiments are designed. Typically, only a few salts allow a significant diffusion of
the receptor within the mesophase. The faster the diffusion rates for the protein in a
set of conditions, the greater the chances of growing crystals under those conditions.
For laboratories with no prior mesophase screening experience, we advise undertak-
ing FRAP measurements and LCP setups using
2-AR(E122W)/T4L or human
adenosine A
2a
receptor (PDB ID 4EIY) samples, which reproducibly crystallize
without the need of automation.
For a positive control in the diffusion assays, 0.2 M sodium citrate, 28% PEG
400, 0.1 M HEPES, pH 7.5, with carazolol-bound
b
2-AR can be used, with 0.5 M
NaCl employed as a negative control (
Xu et al., 2011
). Conditions where we ob-
served crystallization of
b
2-AR (E122W)/T4L that correlate with diffusion rates in-
cluded one of the following salts: ammonium fluoride, dibasic ammonium
phosphate, potassium sulfate, potassium thiocyanate, dibasic sodium phosphate
dihydrate, or sodium sulfate. A screen of additives added to the salts in the preceding
text (or mixture of salts) and PEG 400 after FRAP assays identified the following
compounds as beneficial for the crystallization of
b
b
2-AR(E122W)/T4L: ethylene
glycol, 1,6-hexanediol, 1,3-butanediol, 2-propanol, 1,4-butanediol,
tert-butanol,
1,3-propanediol, and 1-propanol.
The first crystal hits obtained from initial screens are generally not suitable for
X-ray diffraction. Thus, individual conditions should be expanded by extensive fine
grid screens around the initial conditions. Some initial hits, despite such fine screen-
ing, did not improve, although the use of ammonium fluoride, ammonium phosphate,
and sodium sulfate along with 28-30% PEG 400 and 1,4-butanediol (5-8%) pro-
duced diffraction quality crystals of a 20
m size (
Fig. 24.4
B and C).
The most important variable in growing larger crystals is the monoolein/cholesterol
ratio (cholesterol was varied from 3 to 10 mol%). Usually, adding cholesterol to
monoolein slowed crystal growth from 2 days to 2 weeks.
10
5
m
24.2.2.2.3
Monitoring crystal growth
A detailed observation record should be kept for every crystallization tray, no matter
how small the changes observed. Excellent guidelines were established by pioneers
in the field (
Caffrey & Cherezov, 2009
). An automated plate reader is a good invest-
ment because, as in any crystallization trial, once one starts setting drops, the number
of trials grows exponentially. Many setups show crystal growth over a month but
plates beyond a month tend to dry out. This can be delayed by assuring that the sand-
wich tape has no trapped bubbles, wrapping the plates with aluminum foil, and in-
cubating them at 22
C.
A crossed polarizer-fitted microscope with good quality optics is all one needs to
monitor the crystallization process from mesophase setups (
Fig. 24.4
B and C). How-
ever, other more expensive imaging options are recommended to discriminate be-
tween salt and protein crystals and avoid wasting valuable time and resources
following false leads. For example, microscopes equipped with UV fluorescence
(
Judge, Swift, & Gonzalez, 2005
), Second-order nonlinear optical imaging of chiral
crystals (SONICC) (
Kissick, Gualtieri, Simpson, &Cherezov, 2010
), two-photon ex-
cited UV fluorescence (
Madden, Dewalt, & Simpson, 2011
), or two-photon excited
