10. Cryo loops, cryo tools (e.g., CryoWand and CryoTongs),
cryoprotectants (e.g., ethylene glycol and glycerol), and access
to liquid nitrogen, for handling, manipulating, cryoprotecting,
and flash freezing crystals prior to screening and data
collection.
11. Access to an “in-house” X-ray source or dedicated macromolecular synchrotron beamline for screening crystals and data
acquisition.
12. Crystallographic programs to process data and refine threedimensional structures including the CCP4 suite [5] and
COOT [6, 7] (see Note 4 for a list of useful crystallographic
programs and other resources relevant for RNA–protein
crystallography).
3 Methods
3.1 Tools to Predict
the Fold of RNA
Predicting the fold of RNA and defining its length and composition
for use in RNA–protein crystallization experiments can improve the
likelihood of obtaining RNA–protein complex crystals. However,
modeling RNA structure is hampered by its flexibility that allows it
to adopt a wide variety of secondary and tertiary (threedimensional) motifs. Various computational tools exist to predict
RNA structure (see Note 4) including:
l
RNA BRICKS comprises three-dimensional structure motifs of
RNA including information about their contacts with other
RNA motifs, proteins, metal ions, water molecules, or small
molecule ligands. The database provides structure-quality score
annotations and tools for RNA three-dimensional structure
searches [8].
l
RNA 3D MOTIF ATLAS is a comprehensive and representative
collection of internal and hairpin loop RNA three-dimensional
motifs extracted from representative sets of RNA threedimensional structures [9].
l
RNA FRABASE 2.0 is an engine with database to search for
three-dimensional fragments within three-dimensional RNA
structures using sequence(s) and/or secondary structure(s) as
input [10].
3.2 Modeling RNA–
Protein Interactions
When RNA binds to a protein to form a complex, multiple interactions and conformational changes occur in both the RNA and
protein. There are a variety of methods for macromolecular threedimensional structure prediction that can be applied to RNA–protein complexes. Typically, atomic coordinates of experimentally
determined RNA–protein complexes provide the basis for
426
Andrew P. Turnbull and Xiaoqiu Wu
cryoprotectants (e.g., ethylene glycol and glycerol), and access
to liquid nitrogen, for handling, manipulating, cryoprotecting,
and flash freezing crystals prior to screening and data
collection.
11. Access to an “in-house” X-ray source or dedicated macromolecular synchrotron beamline for screening crystals and data
acquisition.
12. Crystallographic programs to process data and refine threedimensional structures including the CCP4 suite [5] and
COOT [6, 7] (see Note 4 for a list of useful crystallographic
programs and other resources relevant for RNA–protein
crystallography).
3 Methods
3.1 Tools to Predict
the Fold of RNA
Predicting the fold of RNA and defining its length and composition
for use in RNA–protein crystallization experiments can improve the
likelihood of obtaining RNA–protein complex crystals. However,
modeling RNA structure is hampered by its flexibility that allows it
to adopt a wide variety of secondary and tertiary (threedimensional) motifs. Various computational tools exist to predict
RNA structure (see Note 4) including:
l
RNA BRICKS comprises three-dimensional structure motifs of
RNA including information about their contacts with other
RNA motifs, proteins, metal ions, water molecules, or small
molecule ligands. The database provides structure-quality score
annotations and tools for RNA three-dimensional structure
searches [8].
l
RNA 3D MOTIF ATLAS is a comprehensive and representative
collection of internal and hairpin loop RNA three-dimensional
motifs extracted from representative sets of RNA threedimensional structures [9].
l
RNA FRABASE 2.0 is an engine with database to search for
three-dimensional fragments within three-dimensional RNA
structures using sequence(s) and/or secondary structure(s) as
input [10].
3.2 Modeling RNA–
Protein Interactions
When RNA binds to a protein to form a complex, multiple interactions and conformational changes occur in both the RNA and
protein. There are a variety of methods for macromolecular threedimensional structure prediction that can be applied to RNA–protein complexes. Typically, atomic coordinates of experimentally
determined RNA–protein complexes provide the basis for
426
Andrew P. Turnbull and Xiaoqiu Wu