Hello,
I have 110 kDa protein along with RNA. I want to perform heterogeneous refinement, I have extracted particle with 320 Box size, So what range of refinement box size is recommended? Because by default it is 128 but I am not sure about it. Pixel size is 0.85 A, 105 K magnification and bining 2.
Next question is my homogenous refinement stop after 3,46 and curves are bit different. I have attached the image of the curves. It will be great if I get guidance for both the questions.
This depends on the kind of heterogeneity you are trying to resolve. If you are trying to eliminate junk particles, then calculate the voxel size for ~20 angstroms max resolution or so (max resolution = 2X apix). If you are trying to separate different conformations, this can be set to ~12 or 8 angstroms. There is no use in including high resolution information at this point, it can be even deleterious - at least IMO.
Because by default it is 128 but I am not sure about it. Pixel size is 0.85 A, 105 K magnification and bining 2.
Next question is my homogenous refinement stop after 3,46 and curves are bit different. I have attached the image of the curves. It will be great if I get guidance for both the questions.
The GSFSC from homogeneous refinement could be approaching Nyquist or the box size is cutting stuff out. ~110kDa, 320 pix, 0.85A | 320pix*0.85A/pix = 272A, 272A should be ~2x the size of your max diameter so maybe the box size is fine. Could experiment.
I do like multi-class ab-initio followed by heterogeneous refinement, but never increased the down sampling to 128pix for hetero - just because it takes so long. Most 3D classification jobs will work best without the high frequency information in the 6-20A range.
Thank you.
This homogenous from initial ab-initio where I have applied Fourier crop (360 Box size, 180 Fourier crop size)
I have run the 2D classes without Fourier crop. Will do ab-initio and homogeneous refinement respectively. Hopefully it should not approach Nyquist.
Good luck @nikhil, the FSCs getting close to the right side was the only obvious thing. Hope it improves things, the RNA you have should be helpful for contrast.
