Crystallizing RNA–protein complexes is particularly challenging
and can be hampered by RNase contamination, which can degrade
RNA, the conformational flexibility of the protein and the inherent
flexibility and negative charge of RNA. In addition, in cases where
the RNA–protein complex comprises multiple subunits, the
increased complexity can make it difficult to structurally characterize the complex. This chapter focuses on methods that can be used
to overcome the unique problems faced when determining the
three-dimensional structures of RNA–protein complexes using
X-ray crystallography.
2 Materials
1. RNase-free stock reagents (see Note 1).
2. Ambion RNaseAlert
® lab test kit to check buffers, RNA, and
protein preps for RNase contamination (see Subheading 3.3).
3. Synthetic or in vitro transcribed RNA (see Subheading 3.4).
4. Purified, crystallization-grade protein (see Subheading 3.7).
Protein can be produced in house, e.g., overexpressed in bacteria, insect, or mammalian cell systems, and purified using,
e.g., an A ¨ KTA protein purifier (GE Healthcare Life Sciences),
obtained through a collaboration with an academic group or
outsourced to a contract research organization (CRO) as a
fee-for-service.
5. Spectrophotometer (e.g., Thermo Scientific™ NanoDrop™ or
Perkin Elmer LAMBDA™ 465) to accurately determine RNA
and protein concentrations and to ensure that protein samples
are RNA-free (see Subheading 3.5).
6. Temperature-controlled, vibration-free crystallization incubator or temperature-controlled room to maintain a constant
temperature for growing crystals during the time course of
the crystallization experiment.
7. Crystal screening reagents including sparse matrix screening
kits (see Note 2).
8. 24-well Linbro plates and siliconized glass coverslips or 96-well
Society for Biomedical Sciences (SBS) format crystallization
plates (e.g., Swissci crystallization plates, which feature conical
wells with a lens effect for improved well visualization and
easier crystal retrieval; https://www.swissci.com/), with access
to a crystallization robot for automated plate preparation (see
Note 3).
9. Cold light source stereo microscope to manually inspect crystallization plates or access to an automated crystallization
imager with plate hotel (e.g., Formulatrix Rock Imager
® ).
Studying RNA–Protein Complexes Using X-Ray Crystallography
425
and can be hampered by RNase contamination, which can degrade
RNA, the conformational flexibility of the protein and the inherent
flexibility and negative charge of RNA. In addition, in cases where
the RNA–protein complex comprises multiple subunits, the
increased complexity can make it difficult to structurally characterize the complex. This chapter focuses on methods that can be used
to overcome the unique problems faced when determining the
three-dimensional structures of RNA–protein complexes using
X-ray crystallography.
2 Materials
1. RNase-free stock reagents (see Note 1).
2. Ambion RNaseAlert
® lab test kit to check buffers, RNA, and
protein preps for RNase contamination (see Subheading 3.3).
3. Synthetic or in vitro transcribed RNA (see Subheading 3.4).
4. Purified, crystallization-grade protein (see Subheading 3.7).
Protein can be produced in house, e.g., overexpressed in bacteria, insect, or mammalian cell systems, and purified using,
e.g., an A ¨ KTA protein purifier (GE Healthcare Life Sciences),
obtained through a collaboration with an academic group or
outsourced to a contract research organization (CRO) as a
fee-for-service.
5. Spectrophotometer (e.g., Thermo Scientific™ NanoDrop™ or
Perkin Elmer LAMBDA™ 465) to accurately determine RNA
and protein concentrations and to ensure that protein samples
are RNA-free (see Subheading 3.5).
6. Temperature-controlled, vibration-free crystallization incubator or temperature-controlled room to maintain a constant
temperature for growing crystals during the time course of
the crystallization experiment.
7. Crystal screening reagents including sparse matrix screening
kits (see Note 2).
8. 24-well Linbro plates and siliconized glass coverslips or 96-well
Society for Biomedical Sciences (SBS) format crystallization
plates (e.g., Swissci crystallization plates, which feature conical
wells with a lens effect for improved well visualization and
easier crystal retrieval; https://www.swissci.com/), with access
to a crystallization robot for automated plate preparation (see
Note 3).
9. Cold light source stereo microscope to manually inspect crystallization plates or access to an automated crystallization
imager with plate hotel (e.g., Formulatrix Rock Imager
® ).
Studying RNA–Protein Complexes Using X-Ray Crystallography
425