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visible fluorescence combined with second harmonic generation (
Padayatti,
Palczewska, Sun, Palczewski, & Salom, 2012
) can help in this regard.
The first breakthrough in any mesophase crystallization trial is obtaining the ini-
tial microcrystals, no matter how small or insignificant they may appear. The next
follow-up steps are to reproducibly grow microcrystals followed by optimization to
obtain larger crystals. Crystal sizes as small as 10
m are adequate for X-ray
diffraction data collection at modern beamlines equipped with microfocus and raster-
ing capabilities (we used beamlines 23-ID (GMC-A) and 24-ID (NE-CAT) at APS,
Argonne, IL). Moreover, the promise of free-electron laser techniques in collecting
data from protein nanocrystals holds great hope for the future (
Dilanian, Streltsov,
Quiney, & Nugent, 2013; Kang, Lee, & Drew, 2013
).
We collected a full data set from a single
5
2
m
2-AR(E122W)/T4L crystal. X-ray dif-
fraction data are shown in
Fig. 24.4
D, with spots extending down to 3.4
˚
.The
resulting molecular replacement solution, along with the corresponding electron
density map, is shown in
Fig. 24.4
E.
b
24.3
DISCUSSION
We have described two very different strategies for GPCR crystallization that can be
adapted to the crystallization of other GPCRs.
Rhodopsin, being a stable and abundant receptor, can be obtained from native
sources and most of the experimental procedures can be performed at room temper-
ature. Trigonal crystals grow very reproducibly in a classic vapor-diffusion format.
The most challenging requirement is the need to work in a dark room under dim red
light.
After the crystal structure of rhodopsin was initially solved, it took 7 years to
solve the structure of the first GPCR with a diffusible ligand,
2-AR/T4L
(
Cherezov et al., 2007
). This was enabled mainly by advances in protein engineering,
crystallization methods, and X-ray collection, which allowed solving the structures
of at least 17 more GPCRs in the following 6 years. In addition, sequence modifi-
cations engineered into the
b
2-AR to enhance its crystallization can also be used
as a reference, although they are often not directly transferable to other GPCRs.
Therefore, before attempting the crystallization of a novel GPCR target, we recom-
mend that multiple protein constructs be subjected to several “stress tests” (thermo-
stability assays, expression level assays, homogeneity determination by gel filtration,
purification yields, radioligand binding, FRAP assays, etc.) before the three to four
best constructs are selected for crystallization trials.
b
Acknowledgments
The X-ray diffraction data were collected at 24-ID-C (NE-CAT) beamline at APS, Chicago.
The authors thank Wenyu Sun for help with insect cell cultures and Philip Kiser for diffraction
data collection.
