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| In EMAN2 an initial model can be constructed using RCT and a stack of untilted and tilted particles whose set element relationship is on-to-one. Typically the tilt angle between the unilted and tilted is at least 45 degrees, but usually 60 degrees. A larger tilt angle is desireable because RCT produces reconstructions with a missing cone, in Fourier space, and a larger tilt angle reduces the cone volume. In some cases -45 and 45 degree tilt data are collected to remove the missing cone altogether. To do a RCT reconstruction in EMAN2: | In EMAN2 an initial model can be constructed using RCT and a stack of untilted and tilted particles whose set element relationship is on-to-one. Typically the tilt angle between the untilted and tilted is at least 45 degrees, but usually 60 degrees. A larger tilt angle is desirable because RCT produces reconstructions with a missing cone, in Fourier space, a larger tilt angle reduces the cone volume. In some cases -45 and 45 degree tilt data are collected to remove the missing cone altogether. For more information see: http://www.oup.com/us/catalog/general/subject/LifeSciences/MolecularCellBiology/?view=usa&ci=9780195182187 To do a RCT reconstruction in EMAN2: |
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| 1. Run: e2refine2d.py on the untilted particle stack. Examine the class averages, and note the rubbish ones. These will be excluded from the rct step. 1. Run: e2rct.py using the data and options described below. |
1. Run: e2refine2d.py on the untilted particle stack. Examine the class averages, and note the rubbish ones. These will be excluded from the RCT step. 1. Run: e2rct.py using the the untilted data, tilted data, class average data, stagetilt and any desired options. |
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| e2rct.py ''options'' | === Using e2rct.py === To run e2rct.py the following options are required: --untiltedata, --tiltdata, --classavg, --stagetilt |
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| Where ''options'' are: | For example: e2rct.py --untiltdata="bdb:RCTparticles#myuntiltedparticles" --tiltdata="bdb:RCTparticles#mytiltedparticles" --classavg="bdb:r2d_01#classes_06" --stagetilt=50 e2rct.py writes out a RCT reconstruction for each class average, and and averaged RCT recon if the option, --avgrcts, is activated. In addition, if the option, --align, is chosen, then the centered tilted particles will be written out. Usually these output files are written to the database. The e2rct.py'''' ''''''options'' are: |
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| * --classavg: The stack of classaverages created by e2refine2d.py (usually this will be in the database) | * --classavg: The stack of class averages created by e2refine2d.py (usually this will be in the database) |
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| * --careject: A comma delimted list of class averages to reject * --align: Center the tilted images before RCT reconstruction (impoves recon quality) (Boolean toggle switch) |
* --careject: A comma delimited list of class averages to reject * --align: Center the tilted images before RCT reconstruction (improves recon quality) (Boolean toggle switch) |
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| * --avgrcts: Align in 3D and average each of the RCT recons, from each classaverage (Boolean toggle switch) * --preprocess: Preprocess the RCT recons before 3D alignment usign an EMAN2 processor * --alignran: Finess of the 3D alignment search |
* --avgrcts: Align in 3D and average each of the RCT recons, from each class average (Boolean toggle switch) * --preprocess: Preprocess the RCT recons before 3D alignment using an EMAN2 processor * --alignran: Fineness of the 3D alignment search |
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| e2rct.py reads in the class averages from e2refine2d.py and extracts the particles making up each class average. The in plane rotation for each untilted particle in each class is used in conjuction with the stage tilt, and optionally tiltaxis, to insert the tilted particle into the RCT recon, after optional centering. The net result is a RCT recon for each class average. These RCT recons are then, optioanlly preprocessed and aligned, and averaged. | e2rct.py reads in the class averages from e2refine2d.py and extracts the particles making up each class average. The in plane rotation for each untilted particle in each class is used in conjunction with the stage tilt, and optionally tiltaxis, to insert the tilted particle into the RCT recon, after optional centering. The net result is a RCT recon for each class average. These RCT recons are then, optionally preprocessed and aligned, and averaged. |
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| * Set --minproj to a reasonable level, at least 30. If the number of projection in a RCT recon is too small then the RCT recon will be very poor quality * If 3D alignment is chosen, then preprocess the RCT recon by aggressively lowpass filteration to boost the signal to noise ratio, enabling a decent 3D alignment * Reject any rubbish looking class averages (smeary, not CTF rings, etc). Binning the images into self simiar bins is critical, if the images ina classaverage are random/junk, then your RCT will be random/junk |
* Set --minproj to a reasonable level, at least 30. If the number of projections in a RCT recon is too small then the RCT recon will be very poor quality * If 3D alignment is chosen, then preprocess the RCT recon by aggressively lowpass filtration to boost the signal to noise ratio, enabling a decent 3D alignment * Reject any rubbish looking class averages (smeary, no CTF rings, etc). Binning the images into self similar bins is critical, if the images in a class average are random/junk, then your RCT will be random/junk |
Reconstruction via Random Conical Tilt (RCT)
In EMAN2 an initial model can be constructed using RCT and a stack of untilted and tilted particles whose set element relationship is on-to-one. Typically the tilt angle between the untilted and tilted is at least 45 degrees, but usually 60 degrees. A larger tilt angle is desirable because RCT produces reconstructions with a missing cone, in Fourier space, a larger tilt angle reduces the cone volume. In some cases -45 and 45 degree tilt data are collected to remove the missing cone altogether. For more information see: http://www.oup.com/us/catalog/general/subject/LifeSciences/MolecularCellBiology/?view=usa&ci=9780195182187
To do a RCT reconstruction in EMAN2:
- Pick untilted/tilted particle pairs using e2RCTboxer.py. While WEB/JWEB or DoG tiltpicker can be used, it is recommended to use e2RCTboxer.py ensuring that image attributes will be correct and avoiding file conversion (which can be a bit of a headache)
- Run: e2refine2d.py on the untilted particle stack. Examine the class averages, and note the rubbish ones. These will be excluded from the RCT step.
- Run: e2rct.py using the the untilted data, tilted data, class average data, stagetilt and any desired options.
Using e2rct.py
To run e2rct.py the following options are required: --untiltedata, --tiltdata, --classavg, --stagetilt
For example: e2rct.py --untiltdata="bdb:RCTparticles#myuntiltedparticles" --tiltdata="bdb:RCTparticles#mytiltedparticles" --classavg="bdb:r2d_01#classes_06" --stagetilt=50
e2rct.py writes out a RCT reconstruction for each class average, and and averaged RCT recon if the option, --avgrcts, is activated. In addition, if the option, --align, is chosen, then the centered tilted particles will be written out. Usually these output files are written to the database.
The e2rct.py' options are:
e2rct.py reads in the class averages from e2refine2d.py and extracts the particles making up each class average. The in plane rotation for each untilted particle in each class is used in conjunction with the stage tilt, and optionally tiltaxis, to insert the tilted particle into the RCT recon, after optional centering. The net result is a RCT recon for each class average. These RCT recons are then, optionally preprocessed and aligned, and averaged.
e2rct.py algorithm
Strategies for success