the concentration of the protein and precipitant in the drop
increases and, if the protein becomes supersaturated, crystals may
form. Many laboratories now feature dedicated crystallization
robots, such as the Mosquito
® crystal (SPT Labtech) and NT8
®
(Formulatrix), that can dispense much smaller (nanoliter) volumes
into 96-well SBS standard plates for automated screening of crystallization conditions. Compared with setting up crystallization
experiments by hand, crystallization robots significantly improve
accuracy and reproducibility, and the ability to dispense nanoliter
volumes dramatically reduces protein sample consumption. Furthermore, crystallization robots can be used to rapidly set up commercially available coarse screens covering a broad range of reagents
and screen formulations (see Note 3). For example, JCSG+ and
PACT are widely used 96-condition coarse screens that provide a
useful minimal crystallization screening strategy (see Note 2)
[19]. In addition, the JBScreen Nuc-Pro (Jena Bioscience
GmbH) and Natrix screens (Hampton Research) are designed to
screen for preliminary crystallization conditions of RNA, DNA, and
protein–nucleic acid complexes. A precipitation pattern ranging
from clear to light/heavy precipitate usually indicates that the
RNA–protein complex is at a suitable concentration for crystallization experiments: too dilute samples result in clear drops whereas
too concentrated samples result in amorphous precipitate. The
PCT™ (pre-crystallization test; Hampton Research) provides a
useful means of determining the appropriate sample concentration
to use prior to setting up crystallization experiments (see Note 6).
Crystallization conditions for published RNA–protein complexes as of 2007 were analyzed [16]. In general, RNA–protein
complex crystals tend to grow at a neutral pH ranging between 6.5
and 7.5, with polyethylene glycol (PEG) representing the most
common precipitant. Divalent ions including magnesium, calcium,
and manganese may also be important components for RNA–protein crystallization. For example, magnesium ions are often seen to
interact with the phosphate backbone of RNA in high-resolution
X-ray structures of RNA or RNA–protein complexes. Conditions
containing phosphate buffer and high salt concentrations, which
can bind to and block RNA-binding sites on proteins, should be
avoided since they can perturb RNA–protein complex formation
and crystallization.
3.11 Optimizing
Crystallization
Conditions
Crystallization trays should be inspected periodically under a cold
light source stereo microscope to check for the appearance of
crystalline material or crystals, over a time course ranging from
days to weeks. Preliminary conditions may be identified that produce single crystals that are sufficiently large (typically !50 μm in
size for synchrotron sources or !100 μm for in-house sources) to
be screened and evaluated on an X-ray system for diffraction properties. It may be possible to collect a complete data set from a single
Studying RNA–Protein Complexes Using X-Ray Crystallography
435
increases and, if the protein becomes supersaturated, crystals may
form. Many laboratories now feature dedicated crystallization
robots, such as the Mosquito
® crystal (SPT Labtech) and NT8
®
(Formulatrix), that can dispense much smaller (nanoliter) volumes
into 96-well SBS standard plates for automated screening of crystallization conditions. Compared with setting up crystallization
experiments by hand, crystallization robots significantly improve
accuracy and reproducibility, and the ability to dispense nanoliter
volumes dramatically reduces protein sample consumption. Furthermore, crystallization robots can be used to rapidly set up commercially available coarse screens covering a broad range of reagents
and screen formulations (see Note 3). For example, JCSG+ and
PACT are widely used 96-condition coarse screens that provide a
useful minimal crystallization screening strategy (see Note 2)
[19]. In addition, the JBScreen Nuc-Pro (Jena Bioscience
GmbH) and Natrix screens (Hampton Research) are designed to
screen for preliminary crystallization conditions of RNA, DNA, and
protein–nucleic acid complexes. A precipitation pattern ranging
from clear to light/heavy precipitate usually indicates that the
RNA–protein complex is at a suitable concentration for crystallization experiments: too dilute samples result in clear drops whereas
too concentrated samples result in amorphous precipitate. The
PCT™ (pre-crystallization test; Hampton Research) provides a
useful means of determining the appropriate sample concentration
to use prior to setting up crystallization experiments (see Note 6).
Crystallization conditions for published RNA–protein complexes as of 2007 were analyzed [16]. In general, RNA–protein
complex crystals tend to grow at a neutral pH ranging between 6.5
and 7.5, with polyethylene glycol (PEG) representing the most
common precipitant. Divalent ions including magnesium, calcium,
and manganese may also be important components for RNA–protein crystallization. For example, magnesium ions are often seen to
interact with the phosphate backbone of RNA in high-resolution
X-ray structures of RNA or RNA–protein complexes. Conditions
containing phosphate buffer and high salt concentrations, which
can bind to and block RNA-binding sites on proteins, should be
avoided since they can perturb RNA–protein complex formation
and crystallization.
3.11 Optimizing
Crystallization
Conditions
Crystallization trays should be inspected periodically under a cold
light source stereo microscope to check for the appearance of
crystalline material or crystals, over a time course ranging from
days to weeks. Preliminary conditions may be identified that produce single crystals that are sufficiently large (typically !50 μm in
size for synchrotron sources or !100 μm for in-house sources) to
be screened and evaluated on an X-ray system for diffraction properties. It may be possible to collect a complete data set from a single
Studying RNA–Protein Complexes Using X-Ray Crystallography
435