information management system) on beamlines at Diamond (Didcot, UK), SOLEIL (Paris, France), EMBL (Hamburg, Germany),
and MaxLab (Lund, Sweden) synchrotron sources. Furthermore,
there are tools integrated into ISPyB that allow data to be reprocessed using the data reduction pipeline. For example, images with
significant radiation damage can be excluded from data processing,
the resolution limit of the data set can be adjusted or data can be
reprocessed in an alternative space group.
In co-crystallization experiments with protein and nucleic acids
(DNA and RNA), it is not always clear whether the crystal contains
the intact complex or just a single component. The program
RIBER/DIBER [23] (see Note 4) represents a useful tool to
judge the nucleic acid content of a crystal based on the diffraction
data prior to attempting to determine its three-dimensional structure. The program provides likelihood estimates of all possible
crystal compositions, i.e., protein only, nucleic acid only, or the
complex. RIBER is specifically tuned to detect the presence of
regular RNA stems in macromolecular crystals and can be used
with RNA–protein complex data provided that the resolution
extends to 3 A ˚ .
3.13 Determining
the Structure
The phase problem represents the major hurdle to obtaining a
three-dimensional structure of an RNA–protein complex once a
complete data set has been collected. Commonly used techniques
to solve protein structures include molecular replacement (MR),
heavy-atom derivatization (isomorphous replacement, anomalous
scattering, and anomalous dispersion), and direct methods. MR
represents one of the most powerful phasing techniques and
accounts for over 69% of all X-ray protein structures and 73% of
RNA–protein complex structures deposited in the PDB (as of
March 2019). MR requires a homologous structure for the protein
under investigation, which is used as a search model to calculate
initial estimates of the phases of the new structure. The search
model can either be a homologous structure from the PDB or a
homology model. Automated homology-based modeling servers
include Swiss-model [24] and I-TASSER [25] (see Note 4). In
cases where there are up to four molecules in the asymmetric unit
of the crystal and the search model is structurally similar to the
target protein (30% or higher sequence identity), it is relatively
straightforward to use MR to determine the structure using programs such as PHASER [26]. One of the most widely used strategies for determining novel protein structures with limited or no
sequence identity ( 30%) to structures deposited in the PDB is
selenomethionine incorporation, where methionine is substituted
by selenomethionine in the protein during expression, and allows
structures to be phased using the single-wavelength anomalous
dispersion (SAD) technique. Traditionally, multiple or single isomorphous replacement (MIR or SIR) methods, in which heavy
Studying RNA–Protein Complexes Using X-Ray Crystallography
437
and MaxLab (Lund, Sweden) synchrotron sources. Furthermore,
there are tools integrated into ISPyB that allow data to be reprocessed using the data reduction pipeline. For example, images with
significant radiation damage can be excluded from data processing,
the resolution limit of the data set can be adjusted or data can be
reprocessed in an alternative space group.
In co-crystallization experiments with protein and nucleic acids
(DNA and RNA), it is not always clear whether the crystal contains
the intact complex or just a single component. The program
RIBER/DIBER [23] (see Note 4) represents a useful tool to
judge the nucleic acid content of a crystal based on the diffraction
data prior to attempting to determine its three-dimensional structure. The program provides likelihood estimates of all possible
crystal compositions, i.e., protein only, nucleic acid only, or the
complex. RIBER is specifically tuned to detect the presence of
regular RNA stems in macromolecular crystals and can be used
with RNA–protein complex data provided that the resolution
extends to 3 A ˚ .
3.13 Determining
the Structure
The phase problem represents the major hurdle to obtaining a
three-dimensional structure of an RNA–protein complex once a
complete data set has been collected. Commonly used techniques
to solve protein structures include molecular replacement (MR),
heavy-atom derivatization (isomorphous replacement, anomalous
scattering, and anomalous dispersion), and direct methods. MR
represents one of the most powerful phasing techniques and
accounts for over 69% of all X-ray protein structures and 73% of
RNA–protein complex structures deposited in the PDB (as of
March 2019). MR requires a homologous structure for the protein
under investigation, which is used as a search model to calculate
initial estimates of the phases of the new structure. The search
model can either be a homologous structure from the PDB or a
homology model. Automated homology-based modeling servers
include Swiss-model [24] and I-TASSER [25] (see Note 4). In
cases where there are up to four molecules in the asymmetric unit
of the crystal and the search model is structurally similar to the
target protein (30% or higher sequence identity), it is relatively
straightforward to use MR to determine the structure using programs such as PHASER [26]. One of the most widely used strategies for determining novel protein structures with limited or no
sequence identity ( 30%) to structures deposited in the PDB is
selenomethionine incorporation, where methionine is substituted
by selenomethionine in the protein during expression, and allows
structures to be phased using the single-wavelength anomalous
dispersion (SAD) technique. Traditionally, multiple or single isomorphous replacement (MIR or SIR) methods, in which heavy
Studying RNA–Protein Complexes Using X-Ray Crystallography
437