Subtomogram reconstruction

3D reconstruction of subtomograms from aligned projections

Overview

This section deals with the reconstruction of subtomograms according to the positions stored in a particle list, which can be the result of template matching (see Localization Tutorial) or of interactive localization using a tool like EMAN2 or UCSF Chimera. The reconstruction is performed by weighted backprojection with different reconstruction centers (= centers of particles).

Things you should know to avoid desaster

Take care during the reconstruction process for three things:

• Coordinate system of projections: Projections are aligned based on a coordinate system that is assigned to the gold markers (or any other reference system). Thus, if the coordinate system that is used for alignment of the weighted projections and that used for reconstructing the volume for picking the particles you will end up in a mess, i.e., your subvolumes likely will not include particles.
• Reconstruction center: During reconstruction of the picking volume you might have provided a reconstruction center of your tomogram that deviates from the default value. Another possibility is that you might have cropped your volume after reconstruction because part of it was simply devoid of any interesting stuff. Both, a specific reconstruction center and cropping of the volume lead to a shift of the coordinates in the particle list compared to the coordinates that would result from backprojection of the aligned projections. Thus, offset must be specified or you will end up in Nirvana.
• Binning factor of particle list and projections: Moreover, you must make sure that you interpret weighted projections and the particle list with the correct binning factor: typically, particles will be picked in a downsampled volume and the subtomograms will be reconstructed from full-sized projections to get the subtomogram at its maximum magnification. The correct binning factor interrelating the two must be provided, otherwise you might also end up in nowhere land.
• Make sure that your particle list is not a mix of particles stemming from multiple tomograms. You should be able to tell by checking the Origin attribute in the particle list XML file.

Reconstruct subtomograms using reconstructWB.py

Using the bin/reconstructWB.py script, you can reconstruct a set of subtomograms (or a single tomogram) from weighted and aligned projections by backprojection. As explained above you need to specify the correct parameters to ensure that the coordinates of your particle list and the projections correspond.

The script reconstructWB.py takes the following parameters:

• --projectionDirectory: Directory containing weighted projections
• --particleList: XML particle list
• --size: size of resulting tomogram. The volumes are always cubic.
• --coordinateBinning: optional: the particle list is typically obtained from a binned tomogram. For reconstruction of unbinned subtomograms the binning needs to be inverted - otherwise the reconstructions would be performed at the wrong coordinates.
• --applyWeighting: optional: apply weighting before reconstruction (if projections are not weighted already)
• --recOffset: Volume that is used for particle localization might have been reconstructed with a coordinate offset or it may be cropped (often in z) to remove 'empty' parts. Since the projection coordinate system is still un-altered the information about discarded volume in x,y,z must be provided to perform reconstructions at the correct coordinates.
  

Here is an example:

reconstructWB.py -p pl.xml --projectionDirectory ../reconstructTomo/alignedProjections/ -s 25 -b 1 -o 0,0,192

Here, we reconstruct the subtomograms specified in the partcilelist pl.xml. All particles will be reconstructed into the ../particles folder. Their cube size will be 25 pixels in each dimension. In the previous tomogram reconstruction step, the tomogram was reconstructed to a size of 512,512,128 pixels. The projections stored in ../reconstructTomo/alignedProjections have a size of 512,512, hence the coordinateBinning parameter is set to 1. However, the only really complicated parameter here is the reconstruction offset determined to -o 0,0,192.