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Flexible refinement in EM maps with NCS

This version of the program uses Non-Crystallographic Symmetry, if available. It generates the entire particle from just the monomer coordinates and the NCS operators. Refinement is still performed against phased phased structure factors along Normal Mode directions of low frequency derived from the monomer.
We do also have a version that calculates the normal modes from the entire particle (not on line).
In addition to the PDB file containing the monomer coordinates and the formatted structure factors data files, you must choose:

  • A file containing NCS operators.
  • The resolution limits of the data.
  • The number of modes to use (increasing frequency order).
The engine of refinement is a Conjugate-Gradient algorithm that will optimize the amplitudes of the modes to fit experimental data.

This is intended to be used with (cryo) electron microscopy data, expressed in reciprocal space. For this, you need first to (Fourier) transform the electron density map of electron microscopy data into structure factors (reciprocal space) in your space group, e.g. P1.

See the Documents section on the left for more details on how to invert your map into structure factors (Get Struct. Fact.).

Your email adress: (Recommended, for notification)

Job title: (Recommended, only alphanumerical characters)

PDB file with monomer to refine (max 2400 aa)

File with unit cell and non-crystallographic symmetry operators: Example (O-style)

File with phased structure factors: (max 70,000)

Lowest & highest reflection resolution to use (Å):

First and last normal modes to use: (max 106)

Input data formats

  • The job title is just for your own identification, but note that it will show up in the public job queue (but your results will not be public).

  • The coordinate files should be in PDB format, with only a single structure (no multiple models). Atoms marked with alternate residue flags will be removed. By default the refinement only uses C-alpha atoms, and the refined output will only consist of C-alpha coordinates.

  • The symmetry data is a text file that describes the box and symmetry operators. On the first line there should be six numbers, representing unit cell dimensions (a,b,c) in Ångströms and the cell angles (alpha,beta,gamma) in degrees.
    This is followed by the number of non-crystallographic symmetry operators to be read, which are using the O program format. Each operator is 3x3 matrix representing the rotation, followed by a vector representing the translation. A couple of examples (you should NOT just copy these):

  • The reflection data is a free-format text file with one reflection per line, including the phase. Columns are separated by at least one space, and there are 5 columns per line. The first three are integers corresponding to the h,k,l indices. Column 4 is the amplitude of the reflection and column 5 the phase in degrees. Reflections outside the low/high limits specified by you will be discarded. An example: Fobs_env.data.

  • The refinement is carried out in reciprocal space using normal mode amplitudes. Low-frequency modes offer the advantage to allow for collective and large- amplitude movements. In the order of 10 modes should work well for normal proteins, but you can experiment with higher values, although the execution will be slower. If you choose to include modes 1-6, the program will also attempt rigid-body refinement.
    In any case, you should watch closely the output file which displays some information about possible CA-CA distances violations. If there are too many of these violations, the model should be discarded and another job (with a smaller number of modes) submitted, until this kind of behaviour disappears.

  • The number of modes is usually 7-16 or 7-21. If you want to refine the position and orientation of the model in the map as well, you can use the first 6 modes (i.e. modes 1 to 16 for instance). Their frequency is damped to soften the effect of this refinement using (lambda(k),k=1,6) = lambda(7)/5., as suggested in Hinsen et al., Biophys. J. (2005) 88:818-827.

Help to get the NCS operators

In most cases, the map will be oriented such that the z-axis is the rotation axis, going through the origin.
If you already have a PDB file (starting model) displaying the desired symmetry, you may run the little Fortran program attached to get the NCS operators relating each of the subunits to the first one.
All you have to do is change the size of the blocks (901 in the example attached).
See some Fortran code for details.
WARNING! However, this should only be done if the PDB file has the desired symmetry, which is the one of the map, of course. Problems may arise if the center of the map is not (0., 0., 0.).

If you want to generate the full PDB file from the refined monomer file, you may run the little Fortran program below using the NCS operators obtained above (prompted by the program as a separate file). See some Fortran code for details.

  Marc Delarue http://lorentz.dynstr.pasteur.fr
Page last modified 17:40 May 10, 2017.