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REFINEMENT

    Although RNA_2D3D allows for the independent generation of initial
A and B model 3D structures, only an A model can subsequently be directly refined. This is because the refinement procedure involves first making a B
model copy of the A model, and it is this copy which is is provided controls for its refinement. Upon completion of refining the copy, it replaces the
original A model and is then deleted.

    The refinement controls give interactive access to the general molecular
modeling program TINKER to do enery minimization and dynamics of selected
segments of the model. Segment selection is provided by the items 'Single Strands (LC)' and 'Specified Segments (GC)' of the 'Refine' pulldown menu. 

	The first item enables the step-wise or automatic energy minimization
of all the single stands of the molecule, each of which only interacts with
its local context defined as its bounding nucleotides which are held fixed.
The 'Specified Segments (GC)' item gives the option of doing refinement
in a global contex of a group of segments. The group is defined in three ways:
'picking the members individually', 'all the single strands', or  the 'entire molecule'. The choice of 'all the single strands' results in their being refined in parallel with rest of the molecule held fixed; hence the feature that the single strands can interact with each other and with the rest of the molecule. Picking the members individually provides the opportunity to
concentrate on critical portions of the molecule and refine them in the
global context mode.  This is in contrast with entering the 'subset level',
defining a subset, and then refining it. In the level-dependent mode,
the ends of each of the comprising segments are held fixed and the segments
can only interact with each other, - not with the rest of the molecule.  

	Choosing the 'entire molecule' as the defined group is a short cut
of having to pick it as a single segment.

	Once a segment group is defined there is provided options for the
kind of refinement to be done. These are:  MIN, MIN-MD and MIN-MD_MIN.
In the MIN type, energy minimization is carried out to optional rms
gradient levels of 1.0 or 0.1. In the MIN-MD type, energy minimization
is first carried out to the 1.0 rms gradient leve and then followed
by a 1.0 picosecond dynamics run. The MIN-MD-MIN type is exactly like
the MIN-MD except that it is followed by an energy minization at the
0.1 rms gradient level. To allow for additional refinement without
having to reselect the active segments of interest, there is a 'more
refinement' option for invoking of any the refinement types.  Typically,
a coarse (rms grad 1.0) MIN refinement, if successfully completed, would be
followed by the fine (rms grad 0.1) MIN refinement or by an MIN-MD run, which
in turn could be repeated to increase MD run time beyond 1 picosecond.

    It is important to keep in mind the fact that energy refinment does not
work if there are significant atom overlaps in the initially produced
3D structure. These need to be removed with the segment postioning tools.
Given this preliminary filtering, a refinement session is typically one
of first doing the 'Single Strand (LC)' refinement and then concentrating
on select portions with the 'Specified Segments (GC)' tool. 

    The refinement modes described above are applicable at all levels: MOLECULE, BRANCH and SUBSET.

    Our refinement strategy is designed with the view of obtaining quick,
but fairly accurate results, in line with the exploratory nature of the
program to efficiently search for viable models satisfying user criteria.
Explicit water is not used. Instead they are implicitly incorporated
via the GBSA method which we supplement with Na+ ions strategically placed
to help neutralize the phosphor oxygens.

    Respecting other molecular modeling computations, like annealing
and the finding of conformation transition pathways, we recommend the use
of standard molecular modeling programs, such as the TINKER program that we
use for our refinement.  A pdb or xyz file can be produced as a link to it or other programs. 

    Regarding the use of constraints, other than the freezing of select atom
or residue positions, there is offered the feature of employing hydrogen bonding and dihedral angle constraints as described in the help topics 'hydrogen_bonding' and 'backbone_dihedrals'.

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THE END
