Question: I am having an issue with the eotest and FSC curves. When the particles are low pass filtered, the FSC curve produced by eotest starts to come up sometimes quite significantly, to around 0.5, at about the resolution frequency that the particles were filtered at. Also, the same thing happens when the models are CTF corrected. I have tested the effect of specifying different CTF correction cutoffs on the FSC curve at 20, 30, and 40A. Each time the curve produced by eotest starts to increase just before the cutoff. The same behavior is observed with the particles low passed using different low pass filters. My particles are negatively stained so I need to do a low pass filter for the reconstruction and the CTF correction is to a low resolution. Do you have any suggestions on how to eliminate this behavior? Answer: there are several possible causes for an FSC curve that rises in this way.