Three Dimensional Reconstruction of Single Particle Specimens using Reference Projections With Defocus Groups


This page describes methods for creating a 3D reconstruction from electron micrographs using defocus groups. Once a set of particle images has been obtained, an initial 3D reconstruction is calculated using coarse projection angles. This is followed by refinement, which iteratively adjusts the angles for finer resolution. The CTF is accounted for by computing reconstructions of groups of particles from micrographs with simlar defocus values ("defocus groups"), and CTF-correction is performed when these reconstructions are merged.

An alternative method is available for creating a 3D reconstruction from electron micrographs without defocus groups. The advantages of using defocus groups is as follows. First is that it can readily account for the non-uniform distribution of signal-to-noise in projection data (Penczek, 2012). Second, we find that reconstructions using particle-level CTF-correction sometimes show artifacts when using iterative backprojection methods, such as 'BP RP' or 'BP CG', whereas the use of defocus groups does not present such limitations. However most laboratories have abandoned the use of defocus groups.

In comparison, CTF-correction is applied at the level of windowed particle images offers its own advantages. First, it circumvents one of the approximations when using defocus groups, namely that all particle in a defocus group follow the same CTF profile. At high resolution, where the CTF oscillates more rapidly, this assumption will not hold. Second, parallelization can be more efficient, since groups can be of identical size, independent of the number of particles at each defocus. Third, particles from what would be sparsely populated defocus groups need not be thrown out. Lastly, interoperability with other software packages which correct for the CTF at the level of particle images will be more straightforward.

The procedures are described in greater detail below.


Contrast Transfer Function (CTF) Estimation
The CTF is estimated for each micrograph, and the micrographs are sorted into defocus groups.
Particle Picking and Selection
Identifies likely particles from the micrographs, and extracts windowed images of individual particles from the micrographs.
The particle images are aligned to reference projections using shifts and rotations.
The averages of each reference view are computed, to assess the distribution of projection views.
3D Reconstruction
An initial volume is calculated by back projection, along with an estimate of its resolution.
Initial alignments are iteratively improved, as each projection is given a chance "to find a better home" in terms of its orientation and phase origin, thus improving the resolution of the initial reconstruction.
Additional methods
Other useful methods, such as classification, difference maps, and frequency correction by X-ray scattering. Additional details about these methods may be found at the techniques page.

Links to further information

Running SPIDER procedure files

In the sections below, various file types are denoted by different fonts:

There are two different choices for running SPIDER procedures: Using SPIRE or using SPIDERprocedures directly.

  1. If you are using SPIRE, then read how to run SPIDER procedure files in SPIRE. SPIRE will create the necessary top level directory.
  2. If you are running SPIDER at the Unix prompt the processing steps are carried out by procedure files, which run many SPIDER operations automatically. To use a procedure in this document, click the procedure filename and copy the procedure to your current working directory. At the beginning of each procedure, there is a list of parameters that can be adjusted according to the particular project. Some of the procedures will call additional procedure(s) (listed below the procedure filename). You need not change anything in the procedures. Use the following format to run a SPIDER procedure:
    spider spi/dat @proc
    where spi is the procedure file extension, dat is the project data file extension, and proc.spi is the procedure file.

The data below are sometimes shown plotted with gnuplot. See the Gnuplot manual

After each SPIRE step or after running each procedure file, check the output files to make sure the results are sensible. If you are not sure what is "sensible", ask an expert.

Creating new reconstruction project

At the start of a reconstruction project, a project directory should be set up with the proper subdirectories and procedure files.

These procedures should be run in the project directory.

Selecting micrographs

Generate a list of micrographs and screen the micrographs.

These procedures should be run in the Micrographs/ directory.

Contrast Transfer Function estimation

Estimate the defocus of each micrograph by calculating its power spectrum. Then group the micrographs into groups of similar defocus.

These procedures should be run in the Power_Spectra directory.

For more information, see;

Particle Windowing and Initial Verification

A particle picking procedure file analyzes each micrograph, cutting out small windows of likely particle candidates. This is followed by a manual selection process that identifies the good particle images and rejects the bad ones. Particles automatically output by the procedure file are said to be windowed; the subset that are manually chosen are said to be verified.

These procedures should be run in the Particles/ directory.


Reference images are generated from the reference volume. Data particles are compared to each reference to find the best match, and the corresponding transformations (shifts and rotations) are written to a doc file. Finally, the transformations are applied to the data images, aligning them to the references.

These procedures should be run in the Alignment directory.

Compute Averages

For all projections, all aligned particles of a given reference view are averaged together. Further particle selection is made by selecting a correlation cutoff threshold to reject some particles. The distribution of particles among projections can be displayed.

These procedures should be run in the Reconstruction directory.

3D Reconstruction

Use the selected aligned particles to create an initial 3D volume. To estimate the resolution of the resulting structure, the particle images are split into two equal sets, and the two resulting reconstruction volumes are compared.

These procedures should be run in the Reconstruction directory.


Refinement essentially performs the reconstruction steps repeatedly, decreasing the angular resolution of reference projections with each iteration, thereby giving the data particles a chance to find a better approximating reference match each time. Thus the data particles are allowed to "settle in" and find better fitting angles, than the initial choices. Refinement is a computationally expensive operation. Before starting a refinement, check the results of the above reconstructions, to ensure that all defocus groups have reasonable particle volumes. Remove any groups that are defective.

These procedures should be run in the Refinement directory.