experiment, may also crystallize. In cases where the crystal comprises only RNA, MR using, e.g., PHASER [26], and the protein as
the search model will fail. The program RIBER/DIBER [23] (see
Note 4) can be used to estimate possible crystal compositions after
data collection, i.e., protein only, nucleic acid only, or complex, to
confirm that it is likely to comprise the intact RNA–protein complex prior to structure determination. When the crystal does not
correspond to the intact complex, any additional crystals that have
been observed in different coarse screen crystallization conditions
should be evaluated and collected on an X-ray source. A crystal that
belongs to a different space group or which has different unit cell
parameters may correspond to the intact complex. If no other
crystals are available to assess, it may be necessary to repeat the
crystallization experiments using a higher affinity RNA substrate
(as determined using biophysical techniques) to prevent complex
dissociation. By following the procedures outlined in this chapter
and carefully considering the design of the RBP/RBD constructs
and RNA, and confirming that the RNA and protein form a complex prior to setting up the crystallization experiments, the likelihood of obtaining crystals corresponding to the complex and
subsequently determining its three-dimensional structure will be
vastly improved.
4 Notes
1. The best way to prepare RNase-free solutions is to start with
initial components that are supplied by vendors RNase-free and
to use preparation techniques that prevent any possible RNase
contamination. For example, whenever possible, RNase-free
plasticware should be used. Stock solutions should be prepared
using 0.22–0.45 μm-filtered deionized water, which should be
RNase-free.
2. Sparse matrix coarse screens are used to scout for initial crystallization conditions. Commercial screens such as PACT and
JCSG+ [19] are available from vendors including Molecular
Dimensions Ltd. (Sheffield, UK), Jena Bioscience (Jena, Germany), and Hampton Research (Aliso Viejo, CA, USA).
3. Crystallization robots such as the SPT Labtech Mosquito
®
crystal and Formulatrix NT8
® drop setter can be used for
setting up crystallization trays in 96-well SBS format. Once
preliminary crystallization conditions have been identified, it
may be necessary to design follow up optimization screens to
improve crystal quality. Access to liquid handling robots such as
the SPT Labtech Dragonfly
® can greatly speed up the optimization process.
442
Andrew P. Turnbull and Xiaoqiu Wu
the search model will fail. The program RIBER/DIBER [23] (see
Note 4) can be used to estimate possible crystal compositions after
data collection, i.e., protein only, nucleic acid only, or complex, to
confirm that it is likely to comprise the intact RNA–protein complex prior to structure determination. When the crystal does not
correspond to the intact complex, any additional crystals that have
been observed in different coarse screen crystallization conditions
should be evaluated and collected on an X-ray source. A crystal that
belongs to a different space group or which has different unit cell
parameters may correspond to the intact complex. If no other
crystals are available to assess, it may be necessary to repeat the
crystallization experiments using a higher affinity RNA substrate
(as determined using biophysical techniques) to prevent complex
dissociation. By following the procedures outlined in this chapter
and carefully considering the design of the RBP/RBD constructs
and RNA, and confirming that the RNA and protein form a complex prior to setting up the crystallization experiments, the likelihood of obtaining crystals corresponding to the complex and
subsequently determining its three-dimensional structure will be
vastly improved.
4 Notes
1. The best way to prepare RNase-free solutions is to start with
initial components that are supplied by vendors RNase-free and
to use preparation techniques that prevent any possible RNase
contamination. For example, whenever possible, RNase-free
plasticware should be used. Stock solutions should be prepared
using 0.22–0.45 μm-filtered deionized water, which should be
RNase-free.
2. Sparse matrix coarse screens are used to scout for initial crystallization conditions. Commercial screens such as PACT and
JCSG+ [19] are available from vendors including Molecular
Dimensions Ltd. (Sheffield, UK), Jena Bioscience (Jena, Germany), and Hampton Research (Aliso Viejo, CA, USA).
3. Crystallization robots such as the SPT Labtech Mosquito
®
crystal and Formulatrix NT8
® drop setter can be used for
setting up crystallization trays in 96-well SBS format. Once
preliminary crystallization conditions have been identified, it
may be necessary to design follow up optimization screens to
improve crystal quality. Access to liquid handling robots such as
the SPT Labtech Dragonfly
® can greatly speed up the optimization process.
442
Andrew P. Turnbull and Xiaoqiu Wu