manual refine

Hi Chao,

What high resolution limit are you setting?  Do you have auto masking set to yes?  And are the stripes all over your reconstruction, or just in some places?

Thanks,

Tim

Hi Chao,

What was your aligment resolution though? not the CC resolution. 3A/4A sounds high for a 4.4A structure?

Tim

Aah I see, well in that case your Signed CC setting will not have an effect. The Signed CC resolution specifies what resolution the signed CC should be used until, after that the unsigned will be used.  In your case, if you specify an alignment resolution of 8A, and a signed CC limit of 3 or 4A then the signed CC limit will have no effect as you are not using frequencies after 8A anyway.  For example, if you put your signed CC resolution to 10A, then the unsigned would be used from 10A-8A.

Micelles do indeed often show bad overfitting.  The way we typically overcome this, is to use a mask, but with filtering instead of true masking.  If you start with a mask that is quite tight to the protein, and does not contain the micelle, import this and select it as the active mask in the manual refinement panel.  Then in expert options under masking - set the "outside weight" to 1.0. Change "Low-pass filter outside of mask" to YES, and then set the resolution to something like 20 or 30A.  This leaves low resolution signal in the reference from the micelle which can actually be useful for alignment, but removes high resolution signal and should therefore prevent overfitting within the micelle.

Cheers,

Tim

There isn't currently a true mask producing function in cisTEM - this will hopefully be added in a future version.  The reason your relion mask isn't working is because it is not tight enough.  Tight masks are bad, but in the case I'm suggesting nothing is truly masked, it is only filtered, so tight masks are not too bad.  One option is to play with the relion settings if that is allowed to produce a tighter mask, filter less, use a smaller or zero cosine edge for example.

Here is how I make my masks, maybe it is helpful.

1. Take your reconstruction and open in chimera - in chimera turn on "hide dust" and adjust the threshold until your structure remains, but the noise and micelle are gone (hopefully this is possible, you may need to play with the sharpening) - you want the lowest value where this is true, record this number.

2. Run make_size_map, it is a command line program included with cisTEM.  when it asks for a binarisation threshold enter the value you figured out above.  This creates a new map where the values for each voxel are replaced with the number of connected voxels.  You should now be able to binarise this program such that it keeps on the density for your protein. You cannot do this in cisTEM currently, you will need to use another program such as EMAN, IMAGIC, TIGRIS etc..  You could use this directly as a very tight mask, but i prefer to dilate it a little bit (maybe a couple of voxels), you can do this by filtering it and rebinarising it for example.

Actually - rereading your post It sounds like you have a map where you have erased the micelle.  You can probably make a good mask from this just by binarising it at the level required to remove the backround and keep your protein (you can check this in chimera).  Again, you need to do this binarisation in another program.

Cheers,

Tim

proc3d should do it :-

proc3d input_map.mrc output_map.mrc bin=threshold_value.

Cheers,

Tim
 

Hi Chao,

If you open the maps in a program like chimera or pymol or other surface renderer, then the co-ordinate of the centre of the map will be taken into account (this is written into the MRC header).  In cisTEM all of this information is ignored and the maps are just considered pixel by pixel.  Thus if you open the two maps in the display that comes with cisTEM (go to Assets->Volumes select the two volumes and click display) you should see that in this slice representation the two line up.

I hope this answers your question?

Tim