EmanProtBoxing

Gregory Sharov edited this page Jul 29, 2016 · 3 revisions

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eman2 - boxer

This protocol is essentially a wrapper around EMAN2 e2boxer.py program. EMAN2 [1] currently provides two algorithms for particle picking: implementation of SWARM [2] and Gaussian based approach (see another protocol) implemented through SPARX (but shared with EMAN2) [3].

The SWARM algorithm is based on template-matching, where the template is a rotational average of several (translationally aligned) manually picked particles. While this usually works for more or less spherical particles, you might have problems picking elongated ones.

The key parameters are particle size, picking threshold (cross-correlation metric) and proximity threshold (minimal distance between particles). Other advanced parameters are quite self-explanatory, you can hover the mouse over any of them in EMAN2 interface to see the help message. EMAN2.1 recommends that the box size to be 1.5–2.5 times larger than the maximum dimension of the particle to perform accurate SSNR (spectral signal to noise ratio) estimations. There is a full discussion of box size issue, and a list of good sizes in the online documentation. However, remember that the box size can be modified during particle extraction.

How to proceed

After you launch the protocol, 4 windows should open:

  • control panel - the main e2boxer window with control parameters

  • micrograph display window - shows the full micrograph

  • particle display window - (with nothing in it) will eventually show the selected particles.

  • micrograph thumbnails - this will only appear if you selected 2 or more micrographs as input. Clicking on one of them will select the current image to be boxed.

The particle picker has 4 modes of operation:

  • Manual - you manually select each particle

  • Erase - to mark large regions of the micrograph which should be excluded from automatic picking

  • Swarm - default semi-automated picking mode. Select a few reference particles, and it will select others. The more references you select, generally the better it will do.

  • Gauss - described elsewhere

Select Swarm picking mode (default), and start by manually selecting 2-3 particles (black boxes). You should see many other boxes appear automatically (green boxes). The more manual references you pick, the better the automatic selection should become. Some particles may cause the picker to become too liberal. In this case, generally picking a few more references manually will help.

  • The Particle Diameter parameter is quite important. This is not the same as box size. If you are having troubles getting good Swarm results, try changing this parameter. You can also change the method to More Selective to get fewer particles.

  • You can delete 'bad particles' by holding down Shift and left-clicking on them. This includes particles that were automatically selected. If you delete one of the references you selected, it will update the auto-picking results.

  • Bad particles or boxes that contain just noise can be damaging to your reconstruction, as they permit noise/model bias to become stronger. It is much better to miss a few good particles if it permits you to exclude obvious 'bad' particles.

  • One caveat to the above, if the good particles that get excluded are all in one orientation, that would be bad

  • When picking particles, it can sometimes help for purposes of more accurately centering and identifying particles, to use a box size smaller than the final size you plan to use for processing. This is absolutely fine. You can use whatever box size you like during the picking process. You can change the final box size to be used for extracting particles in later steps.

Control actions

To pick a particle, use Left mouse button to click on it. You can adjust particle position by clicking on it and dragging the mouse. To remove a particle use Shift + left click. To zoom in and out of the micrograph, use the mouse wheel. To shift/move the micrograph area use Right click + drag.

After you are finished, click on Done button in main e2boxer window, then Scipion window will pop up to confirm whether you want to save the coordinates.

01.e2boxerGUI
Figure 1. GUI interface of e2boxer


References

  • [[[1]]] Tang et al. (2007). EMAN2: an extensible image processing suite for electron microscopy. JSB, 157 (1): 38 - 46.

  • [[[2]]] Woolford D. et al. (2007). SwarmPS: Rapid, semi-automated single particle selection software. JSB, 157 (1): 174 - 188.

  • [[[3]]] Hohn M et al. (2007). SPARX, a new environment for Cryo-EM image processing.. JSB, 157(1): 47 – 55.

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