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Hi, Just to make sure, when you re-ran Polishing, did you do the following? 1. Re-extract particles in the desired box size and pixel size 2. Re-refine them 3. Run polishing Also, what happens if you simply use the default parameters? Best regards, Takanori Nakane On 2018/08/29 18:06, Mark Herzik wrote: > Hi All, > > First, apologies for the long email. > > We have a dataset of a small protein (<150 kDa) collected at a fairly high magnification (0.56 Å/pixel) that we have been trying to process to its fullest using the new Bayesian particle polishing implementation in RELION 3.0. We have hit a proverbial wall in our processing and are hoping some of our fellow EMists could provide some insights as to how best to proceed. > > Implementing Bayesian PP with data that have been binned 2x2 (1.12 Å/pixel) using a 384 pixel box size yields robust particle trajectory alignments, nice dose-weighting diagnostics, and yields a 3D reconstruction that is Nyquist limited (2.3 Å resolution) without CTF refinement. Great. > > However, we are now trying to extract shiny particles with a smaller downsampling (to lower the Nyquist-limiting frequency) and larger box size (to decrease the effects of CTF delocalization, which is quite severe at 0.56 Å/pixel and 200 kV) without much success. > > What we have tried: > 1) Polishing the unbinned data or data binned 1.5x1.5 using parameters trained against those data results in very noisy particle trajectories, a poor B-factor and dose-weighting scheme, and a worse resolving reconstruction as a result of these efforts (~2.6 Å resolution). Quite surprising given that these steps utilize the same particle stack that resulted in the 2.3 Å resolution reconstruction mentioned above. > > 2) Polishing the unbinned data or data binned 1.5x1.5 using parameters trained against the binned 2x2 data yields better results than those obtained in scenario 1 but worse than the binned 2x2 data processing in their entirety (~2.5 Å resolution versus 2.3 Å resolution). > > 3) Training the binned 1.5x1.5 data using a small box size (256 pixels, which aids the training step) and then trying to extract the shiny particles with a larger box size (512 pixels) -> RELION crashes with the following error: > > ERROR: > Box size cannot be changed without re-estimating motion - reference pixel size (0.84 A) is not an integral multiple of movie pixel size (0.56 A)! > > SO we wonder, is there a protocol currently implemented within RELION 3.0 that can use the particle trajectories from downsampled data (the binned 2x2 data in our case) but then extract the shiny particles using a user-inputted pixel size and box size? > > Any and all help will be greatly appreciated. > > Thanks, > -Mark > > Laboratory of Dr. Gabriel Lander > Helen Hay Whitney Foundation Postdoctoral Fellow > The Scripps Research Institute > Integrative Structural and Computational Biology > 10550 N. Torrey Pines Rd. HZ 175L > La Jolla, CA 92037 > Office: (858) 784-9499 > > > ######################################################################## > > To unsubscribe from the CCPEM list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1 > ######################################################################## To unsubscribe from the CCPEM list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1