Refine a structure to high-resolution

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Filling in the GUI

For 3D refinements, select the run-type of 3D reconstruction from the drop-down menu at the top of the GUI.

I/O tab

  • The pixel size (in Angstrom) should be the same as the one used to estimate the CTF parameters.
  • If the reference was not reconstructed from the input images in either XMIPP or RELION, you may assume it is not on the absolute greyscale.
  • Note there are various settings for icosahedral symmetry, also see the Conventions. Make sure your input map is in the one you provide here.

CTF tab

  • CTF-correction is recommended, especially for cryo-EM data. If no CTF correction is to be performed, make sure you phase-flipped your data during preprocessing. See the Prepare input files page.
  • Normalisation correction is robust and therefore recommended in the general case.
  • Intensity correction corrects for distinct grey-scale intensities among the signal in the data, e.g. because due to distinct SNRs among the micrographs. This option is only effective if the data is provided in a STAR file that contains multiple unique strings for the rlnMicrographName label (see the Prepare input files page.

Optimisation tab

  • One typically starts refinement from a medium-low resolution filtered map to minimise model bias. If your input map is not low-pass filtered, it may be filtered internally using the Initial low-pass filter option.
  • Often 25-50 iterations are necessary before the refinement converges to a stable solution, but high-resolution refinement may take even more iterations. A fundamental difference with conventional refinement schemes is that iterating does not only serve to find the optimal orientations, iteration also serves to progressively increase the resolution of the model. Note there is currently no convergence criterion implemented, so the user is responsible for monitoring the convergence, e.g. when the resolution where the rlnSsnrMap drops below one does no longer change.
  • The regularisation parameter determines the relative weight between the experimental data and the prior. Bayes' law dictates it should be 1, but better results are often obtained using slightly higher values (e.g. 2-4), especially when dealing with cryo-data.
  • The particle diameter (in Angstroms) serves to define a soft spherical mask that will be applied to the experimental images to reduce their background noise. If solvent flattening is set to Yes, then also the references will be masked using the same spherical mask (or using a user-provided one under the solvent mask option).

Sampling tab

  • XXXX in development from here on!! XXXX
  • In some cases tilt angle searches may be restricted (e.g. because one knows there are only side views in the data set).
  • Translational search ranges may depend on how well-centered the particles were picked, but often 10 pixels will do the job (translational searches in subsequent iterations are centered at the optimal translation in the previous one, so that particles may "move" much more than the original search range during the course of an entire refinement). Note that pre-centering prior to RELION refinement is not necessary, and also not recommended (it often messes up the Gaussian distribution of origin offsets).

Running tab

  • If available memory becomes a limitation (the main need for memory comes from storing the oversampled Fourier transforms of the references) then running multiple threads on multi-core machines are a good option. Otherwise, MPI parallelisation is probably more efficient.

Continuing an old run

If after completion of a run one decides the refinement had not yet converged, or if one wants to perform additional iterations with for example a finer angular sampling (see below), one can continue an old run by selecting the corresponding option from the drop-down menu at the top of the GUI. The number of iterations of the new run should be higher than the iteration from which to continue, as the iteration numbering will continue from the latter. Note that some options cannot be changed upon continuing an old run, and these are deactivated in the GUI.

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