atoms such as mercury, platinum, or gold are incorporated into the
protein under investigation, can be used to calculate initial phase
estimates provided that the derivatized crystal is isomorphous with
respect to the native crystal. Furthermore, if RNA has been produced using solid-phase synthesis, uracil and cytosine can be substituted with 5
0 -bromo substituents to facilitate phase
determination using multi-wavelength anomalous diffraction
(MAD). The PHENIX [27] software suite is a highly automated
system for macromolecular structure determination that can rapidly
arrive at an initial partial model of a structure without significant
human intervention, given moderate resolution and good
quality data.
3.14 Model Building
Using COOT
COOT is the most widely used crystallographic model building and
validation program and is freely available for both academic and
industrial users (see Note 4). COOT can be used for manual and
automated model building, model completion, and validation
[6, 7]. COOT displays electron density maps and models, e.g.,
protein and RNA, and allows the models to be manipulated to
best-fit the electron density map through real-space refinement.
RNA–protein crystals typically diffract to 2.5 A ˚ resolution or
lower. At this resolution, phosphates, which are electron dense,
and bases, which are large, rigid, and planar, tend to be well
resolved and easy to model whereas other features, e.g., sugar
atoms, can be difficult to accurately position in the electron density
map. In COOT, the interface to build ideal DNA and RNA is
launched by selecting the “Ideal RNA/DNA...” option from the
“Calculate > Other Modelling Tools” dialog. For a given sequence
input by the user, a choice of DNA or RNA, A or B form, single or
double stranded, is presented. Mutation of RNA bases in COOT
can be performed using “Simple Mutate” from the “Calculate >
Model/Fit/Refine. . .” dialog. “Calculate > Model/Fit/Refine. . .
> Add Terminal Residue. . .” can be used to extend the nucleic acid
chain.
RCrane represents a tool for semi-automated RNA model
building into low or intermediate resolution electron density
maps and is incorporated into COOT version 0.7 and above
[28]. RCrane helps the user to place phosphates and bases into
the electron density map and then automatically predicts and builds
the detailed all-atom structure of the traced nucleotides (see Fig. 6).
The RCrane plugin initially produces a low-level trace of the backbone and then calculates the coordinates for all the backbone
atoms. RCrane uses three different means of analyzing the RNA
structure: backbone conformer suites defined using approximate
torsion values, RNA pseudo-torsions that simplify the RNA backbone, and base-phosphate perpendicular (Pperp) distance to determine sugar pucker. The pseudo-torsion and Pperp distance
information are used to predict and build the appropriate
conformer.
438
Andrew P. Turnbull and Xiaoqiu Wu
protein under investigation, can be used to calculate initial phase
estimates provided that the derivatized crystal is isomorphous with
respect to the native crystal. Furthermore, if RNA has been produced using solid-phase synthesis, uracil and cytosine can be substituted with 5
0 -bromo substituents to facilitate phase
determination using multi-wavelength anomalous diffraction
(MAD). The PHENIX [27] software suite is a highly automated
system for macromolecular structure determination that can rapidly
arrive at an initial partial model of a structure without significant
human intervention, given moderate resolution and good
quality data.
3.14 Model Building
Using COOT
COOT is the most widely used crystallographic model building and
validation program and is freely available for both academic and
industrial users (see Note 4). COOT can be used for manual and
automated model building, model completion, and validation
[6, 7]. COOT displays electron density maps and models, e.g.,
protein and RNA, and allows the models to be manipulated to
best-fit the electron density map through real-space refinement.
RNA–protein crystals typically diffract to 2.5 A ˚ resolution or
lower. At this resolution, phosphates, which are electron dense,
and bases, which are large, rigid, and planar, tend to be well
resolved and easy to model whereas other features, e.g., sugar
atoms, can be difficult to accurately position in the electron density
map. In COOT, the interface to build ideal DNA and RNA is
launched by selecting the “Ideal RNA/DNA...” option from the
“Calculate > Other Modelling Tools” dialog. For a given sequence
input by the user, a choice of DNA or RNA, A or B form, single or
double stranded, is presented. Mutation of RNA bases in COOT
can be performed using “Simple Mutate” from the “Calculate >
Model/Fit/Refine. . .” dialog. “Calculate > Model/Fit/Refine. . .
> Add Terminal Residue. . .” can be used to extend the nucleic acid
chain.
RCrane represents a tool for semi-automated RNA model
building into low or intermediate resolution electron density
maps and is incorporated into COOT version 0.7 and above
[28]. RCrane helps the user to place phosphates and bases into
the electron density map and then automatically predicts and builds
the detailed all-atom structure of the traced nucleotides (see Fig. 6).
The RCrane plugin initially produces a low-level trace of the backbone and then calculates the coordinates for all the backbone
atoms. RCrane uses three different means of analyzing the RNA
structure: backbone conformer suites defined using approximate
torsion values, RNA pseudo-torsions that simplify the RNA backbone, and base-phosphate perpendicular (Pperp) distance to determine sugar pucker. The pseudo-torsion and Pperp distance
information are used to predict and build the appropriate
conformer.
438
Andrew P. Turnbull and Xiaoqiu Wu