[3DEM] Handedness determination by Random Conical Tilt
Hong-Wei Wang
hwwang at lbl.gov
Fri Mar 16 15:57:57 PDT 2007
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Dear colleagues,
I have experienced a dilemma in determining the handedness of a single
particle by random conical tilt method. I realized that there must be
something wrong during my reconstruction process because the final
reconstruction volume shows a wrong handedness compared with the available
atomic model strcuture. I am going through the whole process that I did.
Please tell me in which step I might have done wrong to mess up the
handedness determination.
1. I took tilt pair images of the sample in a Tecnai-12 microscope. The tilt
angles were 0 and +55 degrees of the goniometer.
2. The micrographs were recorded on films. I digitized the films in Nikon
Super Coolscan 8000 and converted them to spider format.
3. In WEB interface, I chose the "tilt-particle" command to open the untilt
and tilt micrograph and picked the paired particles. Here I can see that the
two micrographs were both in a portrait orientation with the micrograph
labels at the bottom. The micrograph lables are in a readable orientation.
The tilt axis is about perpendicular to y axis in WEB. I can tell that the
highly defocused area in the tilted micrograph is close to the bottom. After
picking the tilted pairs of particles, WEB calculated out the three angles
of GAMMA, PHI, THETA as about -90, -90, 55 degrees respectively.
4. I did the MSA and MRA of the untilted particles and calculate the
in-plane rotation of each particle in SPIDER as ALPHA. For each class of
untilted particles, I generated the euler angle document file for back
projection reconstrudtion with three columes:
colume one is psi = PHI (output of WEB tilt-pair pick results);
colume two is theta = THETA (output of WEB tilt-pair pick results);
colume three is phi = -ALPHA - GAMMA (ourput of WEB tilt-pair pick
results).
5. I did the back-projection reconstruction of correspondent tilted
particles using BP RP in SPIDER using the angular document file.
My guess is that the micrographs I used for processing are flipped to the
real projection of the single particles. I come up with an explaination as
follows: If I can read the lable on the micrograph in its right order, it
means that I am lying beneath the electron microscope facing up to look at
the specimen. Thus what I see is exactly the mirrored image of the
projection of the sample. In such a case, my reconstruction volume is a
mirror of the real structure.
Please tell me if I am right or wrong. Any comments are very welcome!
Hongwei
-----------------------------------
Hongwei Wang, Ph D
MS 20A-355, LSD, LBNL
1 Cyclotron Rd, Berkeley,
CA 94720
510-642-2222 (O)
510-642-8806 (FAX)
hwwang at lbl.gov
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