We are now ready to move on to running the refinement. Note that you can skip these steps and directly
download the density 1ake_aa_center.xpl and the coordinates
Refining ADK with NMFF
The control program for running NMFF is the perl script nmffem.pl. This script reads the input
parameters that controls the refinement as well as runs the refinement through calls to component programs
that are part of the NMFF suite. The input file is called nmff.inp and contains the following lines:
original pdb = 4ake_aa_rotx20.pdb
target map = 1ake_aa_center.xpl
maximum displacement = 1.0
first mode to use = 1
maximum number of modes = 20
cutoff = 5.5
nmff directory = src/nmff/em
nma directory = src/nma/rtb
resolution = 10.0
In nmff.inp, as shown above, original pdb
corresponds to the atomic structure that is used to start the refinement,
corresponds to the electron density map that is the target of the refinement,
is the variable, in Å, that controls the size of step taken along any normal mode to displace atomic positions in
adjusting the model structure to maximize the correlation between the model and the electron density.
NMFF uses a small set of normal modes (of which there are 3N-6, where N is the number of atoms or sites) as the coordinates
to move the atomic positions to better fit the target electron density. Generally
the first 6 normal modes describe overall translations and rotations of molecule. If we optimally aligned the structure
into the density prior to starting the refinement, e.g., using Situs
and rigid-body translations/rotations, then it is probably not necessary to use these modes during the NMFF
stage. In the present case we purposely rotated the superposed conformation by 20° around the x-axis to
illustrate the combined use of rigid body and collective normal mode refinement and thus we start with
(first mode to use) mode 1.
The line maximum number of modes is the specification of the last
mode to use. Thus for this NMFF refinement we use modes 1 - 20; that is, we only search a 20 variable space to flexibly
fit the structure to the target density.
The line beginning with cutoff specifies the distance cutoff to be used
in constructing the elastic network model for normal mode analysis (see the NMFF papers referenced below). For all atom
refinements, such as we are doing here, a cutoff of 5.5Å is appropriate; for Ca refinement we would
use 8Å (see suggested exercise below). The next two lines indicate where the NMFF electron density map
related (nmff) and normal mode analysis (nma) related programs reside. If these were installed in a specific directory,
e.g., by specifying BINDIR= when you configured and installed NMFF, that path goes here. Finally, we specify
the approximate resolution of the electron density map to which we are flexibly fitting the atomic structure; in the present
example the resolution is 10 Å.
We are now ready to run NMFF and refine the open-state conformation of ADK into the electron density of the closed state.
To do this the unix command given below accomplishes this.
unix>nmffem.pl < nmff.inp
The refinement from this starting configuration, with the all atom model, takes about 1 hour and 10 minutes on an
Intel PIV (3GHz) machine. Thus you may want to focus on the suggested exercise described below if you wish to run the
tutorial in "real time".