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euler angles changed a lot with frealign MODE1

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claire89
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Joined: 11 May 2017

Dear all,
I'm running frealign/v8.09 to do the local refinement and 3D reconstruction for MT structure.
The first 4 rounds are pretty fine,but after the 5th round refinement,the map became much worse.
I checked the .par file,it shows that from round4 to round5, the euler angles changed a lot.
But as follow,the mode I'm using is MODE 1.
I'm wondering have you met this kind of problem before,if yes,what can I do the figure this out?

Thanks a lot,
Claire

round5 .par file

C Date and time 30-07-2017, 14:25 Frealign V8.09 - 06.07.10
C Image format . . . . . . . . . . . . S
C Mode . . . . . . . . . . . . . . . . 1
C Magnification refinement . . . . . . F
C Defocus refinement . . . . . . . . . F
C Astigmatism refinement . . . . . . . F
C Defocus ref. of individual particles F
C Ewald sphere correction. . . . . . . 0
C Beautify the final real space map. . F
C Apply SNR filter final map . . . . . F
C Write out matching projections . . . F
C Calculate FSPR and FSC curves. . . . 0
C Calculate more statistics. . . . . . F
C Padding factor for reference volume. 1
C Outer Radius of object [Angstroms] . 200.00
C Inner Radius of object [Angstroms] . 60.00
C Pixel size [Angstroms] . . . . . . . 1.22100
C % Amplitude contrast . . . . . . . . 0.07
C STD level for 3D mask. . . . . . . . 0.00
C Phase res. / B factor constant . . . 1000.00
C Average phase residual for weighting 0.00
C Symmetry card as input . . . . . . . H
C Helical rotation per subunit . . . -25.71
C Helical rise per subunit . . . . . 8.93
C Number of subunits to average. . . 14
C Number of starts . . . . . . . . . 1
C Stiffness parameter. . . . . . . . 1.00
C First particle . . . . . . . . . . . 1
C Last particle. . . . . . . . . . . . 17176
C Relative magnification . . . . . . . 1.0000
C Densitometer step size (microns) . . 6.4
C Phase residual target. . . . . . . . 90.00
C Phase residual threshold . . . . . . 86.00
C Cs [mm]. . . . . . . . . . . . . . . 2.30
C Voltage [kV] . . . . . . . . . . . . 300.00
C Beam tilt Tx, Ty [mrad]. . . . . . . 0.00 0.00
C Resolution of reconstruction . . . . 2.442
C Low resol. limit refinement. . . . . 200.000
C High resol. limit refinement . . . . 10.000
C Defocus uncertainty. . . . . . . . . 1000.000
C B-factor for parameter refinement. . 0.000
C Input image stack frealign_image_stack.spi
C Input parameter file C_1_frealign_sel_phi_4.par
C Output parameter file C_1_frealign_sel_phi_5.par
C Output shifts file C_1_frealign_sel_phi_5.shft
C 3D reconstruction file C_1_frealign_sel_phi_5.spi
C 3D weights file C_1_frealign_sel_phi_5.wgt
C 3D reconstruction halfset 1 C_1_frealign_sel_phi_5_vol_fsc1.spi
C 3D reconstruction halfset 2 C_1_frealign_sel_phi_5_vol_fsc2.spi
C 3D ave phase residual file C_1_frealign_sel_phi_5_phasediffs
C 3D point spread function C_1_frealign_sel_phi_5_pointspread
C
C PSI THETA PHI SHX SHY MAG FILM DF1 DF2 ANGAST PRESA DPRES
1 342.80 90.95 83.00 0.32 -0.83 52416. 1 23699.2 23475.3 36.91 87.92 20.84
2 319.49 90.49 11.96 1.25 0.63 52416. 1 23699.2 23475.3 36.91 87.76 19.68
3 299.22 91.23 358.32 -1.60 1.15 52416. 1 23699.2 23475.3 36.91 87.72 19.69
4 341.48 92.05 26.61 -0.03 -0.42 52416. 1 23699.2 23475.3 36.91 87.75 19.73
5 295.22 91.93 49.69 0.22 -0.22 52416. 1 23699.2 23475.3 36.91 86.99 18.73
6 299.26 83.42 35.10 -1.06 -0.91 52416. 1 23699.2 23475.3 36.91 86.00 16.18

round4 .par file

C PSI THETA PHI SHX SHY MAG FILM DF1 DF2 ANGAST PRESA DPRES
1 299.31 91.42 326.94 0.69 0.26 52416. 1 23699.2 23475.3 36.91 67.08 -0.04
2 299.08 91.58 327.22 0.65 1.28 52416. 1 23699.2 23475.3 36.91 68.08 -0.01
3 299.22 91.73 327.69 -0.54 -0.07 52416. 1 23699.2 23475.3 36.91 68.03 -0.06
4 299.40 91.74 327.97 0.00 1.67 52416. 1 23699.2 23475.3 36.91 68.02 -0.08
5 299.49 91.79 328.09 -0.11 0.42 52416. 1 23699.2 23475.3 36.91 68.26 -0.16
6 299.57 91.69 328.34 -0.20 0.78 52416. 1 23699.2 23475.3 36.91 69.82 -0.17

niko
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Joined: 5 May 2009
The reason for the large

The reason for the large change is that Frealign imposes constraints on Psi and Theta since they should not vary much from helical segment to helical segment. These restraints are imposed in every round, not just round 4. The strength of the restraints depends on the variability of the angles in the previous round. It looks like you started with a significant variability in round 1 and this variability reduced significantly over three refinement cycles, so the restraints became much stronger. If you want to reduce the restraints, reduce the value for Stiffness.

claire89
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Joined: 11 May 2017
niko, Thanks for your quick

niko, Thanks for your quick reply.

I need to point out clearly...The first par file I listed is from round5 and the second par file is from round4.
So it seems like the 5.par are lack of restrains and I need to give it a higher value for stiffness?

Thanks again,
Claire

niko
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Joined: 5 May 2009
Sorry, I misunderstood. Yes,

Sorry, I misunderstood. Yes, in this case you should increase the Stiffness parameter to something like 50 or 100.