                         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 re-
finement procedure involves first making a B model  copy  of
the  A  model, and it is this copy which is is provided con-
trols 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 mini-
mization 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 con-
text 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  seg-
ments.  The  group  is defined in three ways: 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  mini-
mization  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 ex-
actly like the MIN-MD except that it is followed by an ener-
gy 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  com-
pleted, would be followed by the fine (rms grad 0.1) MIN re-
finement or by an MIN-MD run, which in turn could be repeat-
ed to increase MD run time beyond 1 picosecond.

    It is important to keep in mind the fact that energy re-
finment does not work if there are significant atom overlaps
in the initially produced 3D structure. These need to be re-
moved with the segment postioning tools.  Given this prelim-
inary  filtering,  a  refinement session is typically one of
first doing the ’Single Strand  (LC)’  refinement  and  then
concentrating  on  select  portions with the ’Specified Seg-
ments (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 ob-
taining 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  path-
ways,  we  recommend  the use of standard molecular modeling
programs, such as the TINKER program that we use for our re-
finement.  A pdb or xyz file can be produced as a link to it
or other programs.

    Regarding the use of constraints, other than the  freez-
ing  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_bond-
ing’ and ’backbone_dihedrals’.

                          THE END





