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REFINEMENT

    Although RNA_2D3D allows for the independent generation of two
3D structures, only model A has controls for its refinement.    
One type of refinement
is referred to as 'single-strand' and consists of adjusting its bond lengths and angles
and removing any base-base overlaps.  Another is
called 'tertiary-bond' refinement and consists
of adjusting presribed tertiary bonds to their equilibrium lengths.
In contrast to these two refinement types which involve 'local' energy
minimization, the third type is
global (entire molecule) energy minimization. It can be
done with the option of performing it on the local computer or
a host computer.  The single-strand and tertiary-bond refinements are done
on the local computer.

    When single-strand refinement is invoked, each and every single
strand of the sequence is independently subject to energy minimization.
When selected, a copy of it and its bounding bases is made, and
it is this copy which undergoes minimization under
the constraint that its bounding bases are held fixed. Upon completion
of its minimization, said copy is substituted for the original.
(We note that because a single strand is refined independently of
the rest of the sequence, subject only to the constraint of where
its ends bond to, single-strand refinement can be carried out
very efficiently.)  The program AMBER is used to carry out the
single-strand minimization.

    Single-strand refinement can be done automatically in that it does not
require user intervention, or it can be under user control by stepping
through the single strands one at a time (or skipping if desired).
To aid in visualizing the refinement, a single strand
about to be minimized is colored green, becomes red when undergoing
minimization, and becomes yellow (in the interactive mode) when
its minimization is complete.
As noted above, only Model A can be refined.  But for single-strand
refinement it has proven convenient to use the displaying of a
Model B for monitoring the effect of a Model A refinement.
Thus, upon invoking single-strand refinement, Model A is copied
into model B.  Both are then simultaneously displayed, with model B
superimposed on model A and colored magenta.  The subsequent
refinement, whether automatic or interacive, is done on the copy, model B.
Upon completion of the refinement, optional
user acceptance of the refinement causes model B to be copied into
model A.

    As noted, tertiary-bond refinement is for the purpose of accounting
for tertiary interactions.  Since this normally requires large scale
movements of parts of the molecule, such as an entire stem and its
branches shifting position, there is used a reduced representation
of the molecule.  Thus, only its backbone is used, but subject to constraints
which insure the integrity of the stems (double-helical regions)
and non-overlapping of the bases.  The degrees of freedom consist
of rotations within and at the ends of single strands.  The forces
used to bring the bases participating in a tertiary bond within
hydrogen bonding distance of each other are simple harmonic forces
invoked in a soft manner so as not to overcome the forces used to
preserve stem integrity and prevent overlaps.

    The carrying out of a tertiary-bond refinement in the sense
of a local-type energy minimization requires defining the backbone
segment which is to undergo movement.  It contains one set of ends
of the tertiary bonds while the complementary segment (the fixed part
of the backbone) contains the other set of tertiary bond ends.  In
addition to this fixed-segment definition, it is sometimes useful
to manually rotate this or other segments around the axis passing through
their defining end bases in order to reduce impediments to the
motion of the mobile segment.  Accordingly, these features have been
incorporated in the control menu when tertiary-bond refinement is
selected as the refinement item.  An equivalent control menu is invoked
from the 3D 'edit' pulldown when the 'segment movement' item is selected.

    The forces which are invoked to bring the prescribed tertiary bonds
to their equilibrium values cease to operated when these bond lengths
are within 10% of their equilibrium value.

    Global minimization is normally the last phase of refinement
and is of course optional because of the heavy computing which
may be necessary, depending on the size of the molecule.  The AMBER
program is used for this purpose and may be run on the local
computer or on a host computer with which automatic file transfer
and remote command invokation has previously been established.
An especially written Shell script handles transmitting all the
required AMBER files to the remote computer, starts a Shell script
on the computer to run AMBER on the received files and at the completion
of which automatically transmits the result back.

    As an alternate to the use of AMBER for global refinement, there
is the utility for creating a PDB file via the 'utils' pulldown.
With it one may create a PDB file in either the AMBER format or
BIOSYM format.  The latter is really just a standard type PDB file
supplied with CONECT lines for all the required bonds.  It is designated
as BIOSYM to emphasize that it is indeed acceptable by BIOSYM and that
correct bonding will be insured. 
    
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THE END