[3DEM] Handedness determination by Random Conical Tilt

[Hong-Wei Wang]

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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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