crystallization. The PCT kit comprises four unique, preformulated, sterile filtered reagents. The protein sample is mixed with
the reagents on a glass cover slide and suspended over each
reagent in a sealed well in a Linbro plate, and the drops are
inspected to determine if the protein concentration is suitable
for crystallization screening. If all drops show heavy amorphous
precipitate or are all clear, the protein sample can be diluted or
concentrated, respectively, and the test repeated.
7. RNA–protein complex crystals tend to decay rapidly when
exposed to X-rays; hence, data are collected at cryotemperatures (100 K) in order to reduce radiation damage.
Crystal freezing prevents free radicals that are formed when a
crystal is exposed to X-rays from diffusing through the crystal
and causing secondary radiation damage. A suitable cryoprotectant must be selected in order to prevent ice formation.
Commonly used cryoprotectants include ethylene glycol and
glycerol. A useful method to assess the effectiveness of a cryoprotectant is to mix 2 μL of 100% stock cryoprotectant with
8 μL of the crystallization reservoir solution to give a final
cryoprotectant concentration of 20%, then freeze 2 μL of this
solution in liquid nitrogen in a pipette tip. If the frozen solution appears to be clear in the tip, it should effectively cryoprotect the crystal, whereas if it appears opaque, the cryoprotectant
concentration should either be increased or an alternative cryoprotectant should to be selected.
8. The unipuck holds 16 cryo loops and is compatible with most
sample mounting robots including the BART sample changer
used at the Diamond synchrotron light source, which is capable
of storing 37 unipucks and a total of 592 samples.
Acknowledgments
We would like to thank Dr. Paul Owen at Cancer Research UK
Therapeutic Discovery Laboratories (London, UK) for providing
Fig. 3 and interpreting the data and Dr. Tobias Schmidt at the
Cancer Research UK Beatson Institute (Glasgow, UK) for
providing the data and analysis for Figs. 2 and 4.
References
1. Hentze MW, Castello A, Schwarzl T et al
(2018) A brave new world of RNA-binding
proteins. Nat Rev Mol Cell Biol 19:327–341
2. Rould MA, Perona JJ, So ¨ll D et al (1989)
Structure of E. coli glutaminyl-tRNA
synthetase complexed with tRNA(Gln) and
ATP at 2.8 A resolution. Science
246:1135–1142
3. Berman HM, Battistuz T, Bhat TN et al (2002)
The protein data bank. Acta Crystallogr D Biol
Crystallogr D58:899–907
Studying RNA–Protein Complexes Using X-Ray Crystallography
445
the reagents on a glass cover slide and suspended over each
reagent in a sealed well in a Linbro plate, and the drops are
inspected to determine if the protein concentration is suitable
for crystallization screening. If all drops show heavy amorphous
precipitate or are all clear, the protein sample can be diluted or
concentrated, respectively, and the test repeated.
7. RNA–protein complex crystals tend to decay rapidly when
exposed to X-rays; hence, data are collected at cryotemperatures (100 K) in order to reduce radiation damage.
Crystal freezing prevents free radicals that are formed when a
crystal is exposed to X-rays from diffusing through the crystal
and causing secondary radiation damage. A suitable cryoprotectant must be selected in order to prevent ice formation.
Commonly used cryoprotectants include ethylene glycol and
glycerol. A useful method to assess the effectiveness of a cryoprotectant is to mix 2 μL of 100% stock cryoprotectant with
8 μL of the crystallization reservoir solution to give a final
cryoprotectant concentration of 20%, then freeze 2 μL of this
solution in liquid nitrogen in a pipette tip. If the frozen solution appears to be clear in the tip, it should effectively cryoprotect the crystal, whereas if it appears opaque, the cryoprotectant
concentration should either be increased or an alternative cryoprotectant should to be selected.
8. The unipuck holds 16 cryo loops and is compatible with most
sample mounting robots including the BART sample changer
used at the Diamond synchrotron light source, which is capable
of storing 37 unipucks and a total of 592 samples.
Acknowledgments
We would like to thank Dr. Paul Owen at Cancer Research UK
Therapeutic Discovery Laboratories (London, UK) for providing
Fig. 3 and interpreting the data and Dr. Tobias Schmidt at the
Cancer Research UK Beatson Institute (Glasgow, UK) for
providing the data and analysis for Figs. 2 and 4.
References
1. Hentze MW, Castello A, Schwarzl T et al
(2018) A brave new world of RNA-binding
proteins. Nat Rev Mol Cell Biol 19:327–341
2. Rould MA, Perona JJ, So ¨ll D et al (1989)
Structure of E. coli glutaminyl-tRNA
synthetase complexed with tRNA(Gln) and
ATP at 2.8 A resolution. Science
246:1135–1142
3. Berman HM, Battistuz T, Bhat TN et al (2002)
The protein data bank. Acta Crystallogr D Biol
Crystallogr D58:899–907
Studying RNA–Protein Complexes Using X-Ray Crystallography
445