RNA protein separation

Procedure files and sample data for the separation of a cryo-EM map into nucleic acid and protein densities.

Please note that for the method to work the input volume has to be of fairly high resolution. Between 10 and 15 A should be ok. (If you have a very high resolution (<8 A) you usually can seperate the components just by density thresholding without need for this method.) It is also important that the amplitudes at high frequencies are enhanced to counteract the effect of the envelope function (e.g. by adjusting the power spectrum of the volume to a X-ray scattering curve in solution).

This work is described in A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome Christian M.T. Spahn, Pawel A. Penczek, Ardean Leith, and Joachim Frank. StructureVolume 8, Issue 9, Pages 905-1014 (1 September 2000).

Notes

A 40S ribosome volume was interpolated down to 102 x 102 x102 from which 
histograms and separation were done. 

UPPERMOST PROCEDURE FILE IS: spr.spi

Things that one might want to change in case the results are not OK. Resolution
and pixel size play a role.

1.X45 = 50      ; Cluster size to remove small RNA or protein cluster;

2. X23 = 50     ; Protein and RNA voxels are increased by this percentage to
                ; include solvent
		
3. X32 = 70     ; Minimum size of protein at higher threshold

4. DI
   ??
   ??
   B
   (3) ; Old value was (5), may change this value if PROTEIN histogram doesn't look good
   (5) ;   may change this value if PROTEIN histogram doesn't look good

in SPR_InitSep.spi

5. Look for following comments in SPR_RefSep.spi

   ; NOTE: IF YOU MISS A KNOWN PROTEIN THEN PLANT A SEED AT THE KNOWN PROTEIN LOCATION IN 
   ; SEED150MU???-PROTEIN???.DAT . FOR RNA ONE CAN USE THE SAME TECHNIQUE WITH SEED???MU???-RNA001.DAT

6. Look for following comments in SPR_RefSep.spi

   ; IF 
   ; ERROR IN IDENTITY IS OBVIOUS, SHIFT CLUSTER FROM PROTEIN TO RNA.
   ; NUMBER OF CLUSTER THAT SHALL BE SHIFTED IS INPUTED IN 
   ; p_thc2.spi PROCEDURE WITH RR X55 COMMAND
   
   
7. Might use a value slightly larger value than density threshold at maximum peak of RNA 
histogram for x42. For example, if you see that most of the density is
RNA and not much is left for protein at the end of initial separation then increase
x42 by ~ 10-20 %.
 
 8. If the RNA and Prorein Histogram peaks don't look well separated, then try to
 edit SPR_MakeHist.spi to reflect the following:
 
 
; THRESHOLD THE LOW-PASS FILTERED SPIDER VOLUME TO MAKE A MASK
; NOTE: IF THE HISTOGRAM HAS A SPIKE AT '0' THEN INCREASE THE THRESHOLD VALUE
; BECAUSE MASK IS GETTING OUTSIDE THE VOLUME DENSITY
TH M
_11
output/mask_pro 
B
6 ; This value is changed from 2 to 6


and 

; THRESHOLD THE LOW-PASS FILTERED SPIDER VOLUME TO MAKE A MASK
; NOTE: IF THE HISTOGRAM HAS A SPIKE AT '0' THEN INCREASE THE THRESHOLD VALUE
; BECAUSE MASK IS GETTING OUTSIDE THE VOLUME DENSITY
TH M
_21
output/mask_rna 
B
6 ; This value is changed from 2 to 6


9. If you don't get correct separation you may look at the seeds as they are
   generated sequentially and try to find out which input parameter needs to be
   modified.