RNA protein separation

This directory contains SPIDER 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 102x102x102 voxels from which histograms and separation were done. Interpolated data is in 40s.dat.

The SPIDER procedures used in the seperation:

spr.spi

SPR_MakeHist.spi

SPR_FindHistPer.spi

SPR_InitSep.spi

p_growth1.spi

p_cluster1.spi

p_thc1.spi

SPR_RefSep.spi

p_fun.spi

p_cluster1.spi

p_thc1.spi

p_thc2.spi

p_growth1.spi

p_growth1-0.spi

p_growth1-4.spi

p_growth2-2.spi

SPR_MakeSepVol.spi

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

• X45 = 50      ; Cluster size to remove small RNA or protein cluster;
• X23 = 50     ; Protein and RNA voxels are increased by this percentage to include solvent
• X32 = 70     ; Minimum size of protein at higher threshold
• 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
  

• Look for following comments
  ; 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
  

In SPR_RefSep.spi

• Look for following comments
  ; 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
  
• 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%.

In SPR_MakeHist.spi

• If the RNA and Protein Histogram peaks don't look well separated, 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
  

• 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.