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Flexible refinement in EM maps

Performs refinement of a model against phased phased structure factors along Normal Mode directions of low frequency.
In addition to the PDB and the formatted structure factors data files, you must choose:

  • The crystallographic symmetry operators and cell parameters.
  • The resolution limits of the data.
  • The number of modes to use (increasing frequency order).
  • The cutoff for interactions in the Elastic Network Model.
The engine of refinement is a Conjugate-Gradient algorithm that will optimize the amplitudes of the modes to fit experimental data.

This can also make use of (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 structure to refine (max 2400 aa)
Usually 1 CA atom per residue and/or 2-3 atoms per nucleotide): Ex: PDB

File with unit cell parameters and symmetry operators (see below): Ex: SYMM

File with phased structure factors: (max 20,000)

Lowest & highest reflection resolution to use (Å):

First and last normal modes to use: (max 106)
Careful: Modes # 1-6 are global rot. and. trans. modes

Cutoff (Angstrom) for ENM (increase if there are too many zero frequency modes)

Force constant for adjacent CA atoms (NORMAL or STRONG)

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.
    The format is (6f8.3).
    This is followed by symmetry operators, using the AMoRe program format. Each operator is followed by a '*', and the list is terminated with an 'end' record. 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.

  • The STRONG option for the force constant puts a weight of 1000 to the energy of CA(i)-CA(i+1) distances, compared to 1 for all other (non-bonded) distances (NORMAL option). Only CAs from the same subunit are taken into account. For P(i)-P(i+1) distances (if nucleic acids are present), a weight of 30 is taken for distances between bonded nucleotides.

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