Reconstruction of single particles from
random conical tilt pairs using Pawel's methods
Date 7/30/97 Ramani
Updated 8/7/97 Doryen Bubeck
This procedure is to be used when there is no reference. In the Particle Selection phase
of the procedure the spider formated micrographs are windowed in WEB.
The tilt pairs corrdinates are selected and the corresponding partlices windowed. The following particles
are Aligned using a reference free procedure and categorized through a
heirarchical clustering. Then an Angular file is created and used in an iterative 3d
reconstruction. In the end these reconstructions are merged.
Particle Selection
- 1.
Once the micrographs are digitized, they are converted
from RAW to SPIDER format using b01.raw.
- 2.
The reduced versions of the micrographs are displayed in WEB using the
option TILTED PARTICLES. After looking
for similar patterns (formed by the particles) in the two micrographs
start selecting particles. Each time one selects a particle from the
untilted micrograph, the corresponding pair from the tilted micrograph has
to be selected too.
- 3.After selecting 5-6 pairs fit the angles using
the menu option. This determines the theta, gamma and
phi angle between the two micrographs which correspond to the tilt
angle, the angle between the tilt axis and the y-axis in the untilted
micrograph and the same in the tilted micrograph. Once the angles are
fitted, the program automatically goes to the corresponding tilted
particle when given the untilted one.
- 4.
Readjust the final position of the corresponding tilted particle and keep
fitting the angles sporatically to refine the angular parameters.
- 5.
Create a noise file from the untilted micrograph. Use b02.wnd
to window particles from the original, unreduced, tilted, and
untilted micrographs.
- 6.
In WEB with the CATEGORIZE command is used to
montage the particle image files. Manually clicking on each good
particle and save them in a doc file.
Important things to remember are:
-
When the reduced micrographs are displayed in WEB, display the untilted
micrograph first and then the tilted micrograph next.
-
Before fitting the angles initialize the angles gamma and phi equal to
-90.0 degrees to keep the handedness correct.
-
Make sure that you input the correct number for the reduction size
before starting to pick the particles.
-
Gamma is the angle between the tilt axis and the image y-axis in the
untilted micrograph and phi is the corresponding angle in the tilted
micrograph respectively.
Alignment
- 1.
Create a selection document file having the key equal the number of images that have
been selected and will be used in alignment. b04.sel
- 2.
Perform alignment using AP SR command which does a
reference-free alignment and determines the shifts and the rotation
angles. b05.aps
- 3.
Rotate and shift particles according to the alignment parameters. b06.rts
- 4.
Run the initial part of the correspondence analysis program and then
use the sequential file created here (remember that it has to be in
upper case ) to perform hierarchical clustering on the images using CL
HC. Obtain averages to check on distinct views of the
particle and check the homogeneity of the subgroups of particles
belonging to each average. b07.cas
- 5.
A dendogram option can be used as an alternative in WEB to look at the
averages from each groups. You can also use the option MONTAGE
FROM DOCUMENT FILE in WEB to look at the individual images that
form a subgroup. They are represented by an average showing a
distinct view of the particle. The document files containing the image
numbers of each subgroup are created in the CL HE command.
- 6.
Once satisfied with the averages, procede to the the 3D for each view
Creation of the ANGULAR FILE
and the 3D reconstruction.
- 1.
Create a file containing the image numbers and their corresponding
micrographs to determine the angles gamma and phi and
theta from the dcb*** file. b08.crt
- 2.
Create the angular file using the job b09.ang.
Do a 3D for each view of the particle i.e., from the corresponding
tilted particles belonging to that subgroup. b10.bpr.
- 3.
To refine the reconstruction use a projection alignment
procedure b11.prj. This procedure projects the
volume created and cross correlates the input projections
with the projected images appling the correct shifts. Each
time it creates a new volume from the refined projections.
Merged Reconstruction
- 1.
Determine the orientations between the three volumes and also
check it visually. Use command OR 3Q between a reference
volume and the second (b12.orq). and it gives the
three Eulerian angles between them (phi, theta and psi in that order).
Check the value of the cross-correlation coefficient that it
prints along with the angles to verify that the coefficient is indeed
high.
- 2.
Once you have all the angles determined between the different
volumes then you can figure out whether they cover the missing cone and
if you have all the missing information to cover the missing cone. (the
angle theta, is typically around 90 degrees or higher indicating that
when you would merge the two volumes they would cover the missing cone.
- 3.
Once you have the angles determined between the different volumes,
go back to the original angular file and change those angles
corresponding to the second volume by using VO RA command in b13.vor. Then run
b14.ppg to
insert "rotated" angles into original angular document file
(or run the executable version of the
program pprog.f).
This angular file combined with merged projection series and merged
groups should be used to obtain a merged reconstruction. If you have
three groups then first merge the first two groups and then get the
correct angular file for the two merged groups and then merge the third
one with this corrected angular file so that you get a final merged
angular file.
- 4.
Combine the projection series by combining their image numbers in the
selection document files. b14.grp.
- 5.
b15.mer obtains the merged reconstruction.
- 6.
One can refine the merged reconstruction in the same manner as for
individual reconstructions i.e., similar to b11.prj.
Results
The final reconstruction could be used as a reference for
future projection matching procedures provided it has no missing
angular information. Even if the entire missing angular range is not
covered in the merged reconstruction it will still present a better
reconstruction as it atleast covers a part of the missing angular range
compared to a reconstruction from just a single view. For obtaining
reconstructions from reference projections click here.