visualized in COOT. Use “Previous Suite”/“Next Suite” to
select different nucleotides in the chain.
12. Click “Done” once the conformers have been reviewed. This
will close the RCrane window.
13. The RCrane-traced RNA molecule will appear in the COOT
display manager as a separate molecule. Use “Edit > Merge
Molecules” in COOT to merge the RCrane-traced RNA and
protein into a single molecule.
14. The existing RNA chain can be extended in either the 3
0 or 5
0
direction using the “Extend chain. . .” option from the
RCrane menu.
15. The “Rotamerize existing structure. . .” option from the
RCrane menu can be used to rebuild the RNA structure, e.g.,
to fix sugar pucker errors or steric clashes.
16. The fit of the model of the RNA–protein complex to the
electron density map can be improved using the “Calculate >
Model/Fit/Refine. . . > Real Space Refine Zone” option
in COOT.
3.15 Validating
the RNA Structure
Various software tools exist to validate geometric parameters and
interactions in order to identify errors, e.g., steric clashes, which
need to be fixed during the model building and refinement process.
For example, the MolProbity structure-validation web service
(see Note 4) can be used to analyze the RNA backbone since it
adopts rotameric conformations [29]. MolProbity uses the same
Pperp distance technique as RCrane to check sugar puckers
(!2.9A ˚ ¼ C3
0 -endo pucker; <2.9A ˚ ¼ C2
0 -endo pucker).
3.16 Analyzing
the RNA–Protein
Structure
Following refinement, the RNA–protein interface can be analyzed
to provide insights into structure and function. In addition, the
PRI HotScore web server (see Note 4), which uses in silico alanine
scanning mutagenesis to calculate interaction scores for RNA–protein complexes, can be used to identify potential “hotspots” in the
interface [30] that can be targeted to develop therapeutic agents
against medically important RBPs.
3.17 What To Do
When It Is Not
a Complex. . .
Following crystallization, data collection, and phasing, inspection
of the resultant electron density map may reveal that the data do
not correspond to the intact RNA–protein complex but rather to
an isolated component of the complex, i.e., the RBP, RBD, or
RNA. This can occur if the complex dissociates during the time
course of the crystallization experiment and one or more of the
uncomplexed components crystallizes. In particular, pH can have a
dramatic effect on the affinity and stability of the complex. In
addition, unbound RNA, or RNA that has been degraded due to
RNase contamination during the time course of the crystallization
Studying RNA–Protein Complexes Using X-Ray Crystallography
441
select different nucleotides in the chain.
12. Click “Done” once the conformers have been reviewed. This
will close the RCrane window.
13. The RCrane-traced RNA molecule will appear in the COOT
display manager as a separate molecule. Use “Edit > Merge
Molecules” in COOT to merge the RCrane-traced RNA and
protein into a single molecule.
14. The existing RNA chain can be extended in either the 3
0 or 5
0
direction using the “Extend chain. . .” option from the
RCrane menu.
15. The “Rotamerize existing structure. . .” option from the
RCrane menu can be used to rebuild the RNA structure, e.g.,
to fix sugar pucker errors or steric clashes.
16. The fit of the model of the RNA–protein complex to the
electron density map can be improved using the “Calculate >
Model/Fit/Refine. . . > Real Space Refine Zone” option
in COOT.
3.15 Validating
the RNA Structure
Various software tools exist to validate geometric parameters and
interactions in order to identify errors, e.g., steric clashes, which
need to be fixed during the model building and refinement process.
For example, the MolProbity structure-validation web service
(see Note 4) can be used to analyze the RNA backbone since it
adopts rotameric conformations [29]. MolProbity uses the same
Pperp distance technique as RCrane to check sugar puckers
(!2.9A ˚ ¼ C3
0 -endo pucker; <2.9A ˚ ¼ C2
0 -endo pucker).
3.16 Analyzing
the RNA–Protein
Structure
Following refinement, the RNA–protein interface can be analyzed
to provide insights into structure and function. In addition, the
PRI HotScore web server (see Note 4), which uses in silico alanine
scanning mutagenesis to calculate interaction scores for RNA–protein complexes, can be used to identify potential “hotspots” in the
interface [30] that can be targeted to develop therapeutic agents
against medically important RBPs.
3.17 What To Do
When It Is Not
a Complex. . .
Following crystallization, data collection, and phasing, inspection
of the resultant electron density map may reveal that the data do
not correspond to the intact RNA–protein complex but rather to
an isolated component of the complex, i.e., the RBP, RBD, or
RNA. This can occur if the complex dissociates during the time
course of the crystallization experiment and one or more of the
uncomplexed components crystallizes. In particular, pH can have a
dramatic effect on the affinity and stability of the complex. In
addition, unbound RNA, or RNA that has been degraded due to
RNase contamination during the time course of the crystallization
Studying RNA–Protein Complexes Using X-Ray Crystallography
441