There are at least three steps to a tilt experiment
- Record a tilt-pair of micrographs, where the second view is of the same field as the first, but the specimen was rotated by a known angle (rotation angle) about a known axis (tilt axis).
- Determine orientations (phi, theta, omega or phi, theta, psi, where psi = -omega) and origins and then a reconstruction from the first view. At a minimum, the origins of the particles in the second view must be determined. (The second view can be included in the reconstruction--after orientations and origins are determined of course.) Chiral features should be apparent in the reconstruction. The handedness of this structure is termed "hand A". The mirror of this structure is hand B.
- Compare projections of each handedness to images of the second view. In the Bhandedness program, the following steps are performed:
For a detailed discussion of the handedness-tilt experiment, please see Belnap et al (1997) "A Method for Establishing the Handedness of Biological Macromolecules" J. Struct. Biol. 120, 44-51
- The input three-dimensional, hand A map is inverted through the center to give the hand B map: mapB(x,y,z) = mapA(-x,-y,-z).
- 180 degrees is added to each omega or psi angle. This gives the orientation for hand B.
- Two predicted orientations for the second view are computed from the first orientation and the known tilt-axis direction and rotation angle. One predicted orientation corresponds to the expected second orientation if the structure has handedness A. The other corresponds to the expected second orientation if the structure has handedness B. For the hand A orientation the program uses the original psi or omega orientation. For the hand B orientation the program uses the orientation psi (or omega) + 180 degrees. The effect of this is to rotate opposite directions about the tilt axis--as far as the phi, theta orientation values are concerned.
- The hand A map is projected in the predicted hand-A orientation (for the second view).
- The hand B map is projected in the predicted hand-B orientation (for the second view).
- The hand A and hand B projections are compared to the image of the second view. A correlation coefficient is computed for each comparison. The correct handedness will have the highest correlation coefficient (CC). (Note: Before the test is considered reliable, there should be a significant gap between the hand A and hand B CCs. What is reliable will depend on the specimen and the quality of the images.)
- As a control, the hand A map is projected along the hand A orientation of the first view. This projection is then compared to the first image. This resulting CC should be a similar value to the CC from the correct handedness in part f.
To run dhand you need the following
- Particle images from the first and second views. Particle image numbers must match between the two micrographs, i.e. the id numbers in micrograph pairs must represent the same imaged object. Note, the two images must be kept in the same view direction (i.e. the same x,y orientation) when displaying, storing, extracting, and manipulating in the computer. For example, if you scan negatives you should place the two micrographs in the scanner in the same orientation.
- A three-dimensional reconstruction computed from the micrograph of the first view. (There is no reason the second view cannot be included in the reconstruction as well if the orientations and origins are sufficiently well determined.) For a reliable detection of the correct absolute handedness, the reconstruction needs to be of sufficiently high quality that you can see chiral features (handedness) in it.
- Orientations and origins of particles in the first view, and at least origins of particles in the second view (orientations are okay, too).
- The angle of the tilt axis in the image plane (tilt-axis direction). This angle is measured from the +x-axis of the orientation of the micrograph in the computer (see diagram). A positive angle is a anticlockwise rotation.
- Tilt angles for each view. The tilt angle is the angle of the specimen in the microscope. For example, if the first view was taken with the microscope goniometer at 0 degrees and the second at 5 degrees, then the tilt angles are 0 and 5 for the first and second views, respectively. The angle of rotation is tilt_angle2 - tilt_angle1. It is critical that the tilt and the tilt-axis direction angles be set properly otherwise the incorrect handedness may be chosen (see diagram). For this reason, it is recommended that you use a handedness calibration standard--a particle with known handedness--to calibrate the tilt-axis direction and tilt angles. A standard should be used at every magnification and microscope used since the tilt-axis direction changes with changes in magnification or instrument.
- Radial and resolution limits for the correlation-coefficient calculation, in pixel and angstrom units, respectively.
- Input parameters in a STAR-format file, see example. Multiple micrograph pairs can be entered. The program assumes that successive micrographs (i.e. i and i+1) are pairs.
- The spacing in angstroms/pixel must be set in the header of the input reconstruction and the image files. Use the Bsoft program bhead to set this.
- To run enter the command "dhand [options]
input.star".
The program's help screen is displayed when you enter "dhand" only.
Currently, the program only prints output to the screen. The correlation coefficients are written by default. More output is printed by entering a verbosity number higher than zero. Try "-v 7", for example. The predicted orientations can be output in this way.
Sample output for a test with murine polyomavirus
dhand, version 2001-10-18
Total number of micrographs: 2
Total number of particles: 61
Tilt pair
poly_5122.pif
poly5123.pif
Tilt-axis direction = 113.500 Rotation angle (tilt_angle2 - tilt_angle1) = -5.000
Particle images vs. projections
Correlation Coefficients
projection projection
projection
view 1
view2,handA
view2,handB
Particle_Id
vs. image1 vs. image2
vs.
image2
================================================================
2
0.4206
0.3559
0.1399
3
0.3896
0.3354
0.1670
4
0.4481
0.3817
0.1612
5
0.4178
0.3675
0.1155
6
0.4620
0.3647
0.2844
7
0.4171
0.3803
0.1356
8
0.4146
0.3400
0.1925
9
0.3624
0.3721
0.1674
10
0.4258
0.3838
0.1240
11
0.4803
0.3310
0.1159
12
0.4524
0.4156
0.1735
13
0.4301
0.3405
0.1247
16
0.3839
0.3482
0.0307
17
0.4359
0.3881
0.2008
18
0.3944
0.3550
0.1037
19
0.3994
0.3581
0.1494
22
0.4113
0.3542
0.1109
24
0.4425
0.4033
0.1467
29
0.4380
0.4038
0.1486
Tilt-pair
average
0.4224
0.3673 0.1470
Tilt-pair std. dev
0.0290
0.0245 0.0503
Pair
Count
19
0
Particle images vs. projections
Correlation Coefficients
projection projection
projection
view 1
view2,handA
view2,handB
Particle_Id
vs. image1 vs. image2
vs.
image2
================================================================
Global
average
0.4224
0.3673
0.1470
Global std.
dev
0.0290
0.0245
0.0503
Global
Count
19
0
NOTE: Hand A is handedness of input map.
Mirror of input
map is hand B.
(for multiple micrograph pairs the global results would
represent all
pair-wise comparisons)