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


EC CL           ;;;;;;;;  cluster analysis
dif001
1-75            ; Range of slices to be searched
5               ; Threshold
clr001          ; Output


; Look at output in Web to get 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