Isolating Ligands from 3D Ribosome Reconstructions

Three ways to identify or isolate small masses in 3D reconstructions:

  1. There is a procedure file for constructing difference maps. The statistics of the volumes may be used to determine the significance of 3-dimensional features (see pages 252-254 in Three-Dimensional Electron Microscopy of Macromolecular Assemblies by J. Frank).

  2. The SPIDER operation DR DIFF can be used to compute the error and difference between two volumes.

  3. The set of operations in the procedure below may be entered interactively to extract and isolate a mass from a volume: 

    4. 
; Procedure for obtaining a small isolated mass (e.g., ligand)
; for display purposes. This is not a procedure file, but rather
; a set of routines to be entered interactively.
;
; vol-enhanced-filt = Volume with extra mass
; reference_volume  = Volume without the mass
  

; "Remove" ribosome, i.e., create mask of space around ribosome

TH M
  reference_volume   ; Volume        (input)  
  _1                 ; Volume        (output)
  b                  ; If pixel > threshold: mask = 1, else mask = 0
  0.0012             ; Threshold

NEG                  ; Invert contrast 
  _1                 ; Volume        (input)
  _2                 ; Volume        (output)

MU                   ; Multiply inverted mask * volume with ligand
  vol-enhanced-filt  ; Volume        (input)
  _2                 ; Input volume
  dif001             ; Removed reference ribosome    (output) 
  *                  ; (p > thresh is set to 0


EC CL                ; Cluster analysis
  dif001             ; Volume        (input)
  1-75               ; Range of slices to be searched
  5                  ; Threshold
  clr001             ; Volume        (output)


; Look at output in Web to get density (here 113) of cluster of interest.

TH C 
  clr001              ; Volume        (input)
  _2                  ; Volume        (output)
  113, 10000          ; Replaces density 113 with 10000

TH M
  _2                  ; Volume        (input)
  _3                  ; Volume        (output)
  B
  9999                ; Set all to 0 except the cluster of interest.

FS                    ; File statistics
_3                    ; Volume        (input)

CP                    ; Copy
  _3                  ; Volume        (input)
  clr002              ; Volume        (output)

DI                    ; Dilate - increase size of the cluster
  clr002              ; (segregated cluster will be used as mask) (input)
  clr003              ; Volume        (output)
  B
  5
  5

DI                    ; Dilate
  clr003              ; Volume        (input)
  clr004              ; Volume        (output)
  B
  5
  5

; The next 3 operations remove from the cluster any regions which
; overlap with reference volume.

TH M                  ; Threshold
  reference_volume    ; Volume        (input)
  _1                  ; Volume        (output)
  B
  0.003  

NEG                   ; Invert contrast
  _1                  ; Volume        (input)
  _2                  ; Volume        (output)

MU                    ; Multiply files
  clr004              ; Volume        (input)
  _2                  ; Volume        (output)
  clr005
  *


FS                    ; Get file statistics
  clr005              ; Volume       (input)

MU
  vol-enhanced-filt   ; Volume        (input)
  clr005              ; Volume        (input)
  _2                  ; Volume        (output)
  *

FQ NP                 ; Butterworth low-pass filter              
  _2                  ; Volume        (input)
  vol-isolated        ; Volume        (output)
  7                   ; Filter type
  0.28, 0.38

; Filtered, isolated mass is now in: vol-isolated

Source: isolate.html     Created: 3/29/01     Bill Baxter