Defocus refinement for individual particles

Submitted by donghua on Thu, 2012-01-19 22:00

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Frealign

I am trying Frealign 8.09. The options FPART and DFSIG are for defocus refinement of individual particles. I used FPART=T and DFSIG=150.0, however, the defocus parameters for each particle did not change at all after refinement.
Should I turn on FDEF and/or FASTIG options at the same time in order to refine the defocus values? I remember FDEF and FASTIG can only be done based on each film (the particles from the same film are being refined together).

niko Thu, 2012-01-19 23:20

Yes, you have to set at least FDEF=T for defocus refinement. Setting FPART=F means that the defocus will be refined per micrograph. In other words, the defocus change for all the particles that have the same number in the FILM column in the parameter file will be the same. This should reduce error in the refinement as the signal of all the particles in one micrograph is combined. If one has particles with a very high molecular weight, such as icosahedral viruses, one can set FPART=T. Frealign will then refine the defocus per particle and particles within one micrograph can deviate. The value given for DFSIG will impose some restraint on the defocus refinement, thus helping to prevent unreasonable refined values. The FPART=T option should be used with caution as one can easily end up with defocus values that are worse than the starting values. FASTIG=T will turn on refinement of the astigmatic angle. However, this is almost never justified given the noise level if typical cryo-EM images (even images of viruses). Therefore, FASTIG should be set to F in most cases.