There are several ways to identify or isolate small masses in 3D reconstructions:

• A procedure file for constructing difference maps is on the Single Particle Reconstruction page. 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).

• Alternatively, the SPIDER operation DR DIFF computes the error and difference between two volumes.

• For display and illustration purposes, the set of operations in the recipe below may be entered interactively to extract and isolate a mass from a volume:

; Recipe 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
_1          ; output
b           ; if pixel > threshold: mask = 1, else mask = 0
0.0012          ; threshold

ar
_1
_2
(p1-1)*(-1)     ; inverts mask: ribosome = 0, space = 1

mu          ; multiply inverted mask * volume with ligand
vol-enhanced-filt
_2
dif001          ; removes reference ribosome from volume
*           ; (p > thresh is set to 0

;;;;;;;;  cluster analysis
ec cl
dif001
1-75            ; range of slices to be searched
5           ; threshold
clr001          ; output
;;;;;;;; 

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

th c 
clr001
_2
113,10000       ; replaces density 113 with 10000

th m
_2
_3
b
9999            ; set all to 0 except the cluster of interest.

fs
_3

cp
_3
clr002

di          ; dilation - increase size of the cluster
clr002          ; (segregated cluster will be used as mask)
clr003
b
5
5

di
clr003
clr004
b
5
5

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

th m
reference_volume
_1
b
0.003  

ar
_1
_2
(p1-1)*(-1)

mu
clr004
_2
clr005
*


fs
clr005

mu
vol-enhanced-filt
clr005
_2
*

fq np
_2
vol-isolated
7
0.28,0.38

; Filtered, isolated mass is now in vol-isolated

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