CTF fitting and performance checks with the Volta phase plate

Transkript

1 Max Planck Institute of Biochemistry Martinsried, Germany MAX PLANCK SOCIETY CTF fitting and performance checks with the Volta phase plate Rado Danev currently: Max Planck Institute of Biochemistry, Martinsried, Germany. from April 1 st : The University of Tokyo, Tokyo, Japan March 2018

2 Phase plate TEM Conventional (defocus) Conventional TEM Zernike Phase Plate TEM Hilbert diff specimen aperture objective lens back-focal plane phase plate image forming lenses image

3 TEM imaging modes Conventional TEM CTEM Zernike Phase Plate ZPP Volta Phase Plate VPP central beam thin film central beam hole thin film central beam OL aperture

4 Phase contrast transfer function weak object CTF( k) sin 2 1 z k C 4 s k 4 (k) aberration term phase shift

5 -sin( (k)) cos( (k)) Contrast transfer function; - /2 phase plate conventional TEM phase platetem sin( ( k)) cos( ( k)) 300 kv, = nm, C S = 3 mm 300 kv, = nm, C S = 3 mm 1.0 z = 93 nm z = 56 nm z = 0 nm -1.0 z = 0 nm k [1/nm] k [1/nm]

6 VPP phase shift vs. dose The phase shift increases with the total dose on the phase plate.

7 CTF evolution CTF=sin( (k)) underfocus condition CTF=sin( (k)+ )

8 VPP phase shift evolution 12 nm thick carbon film; beam current: 1.0 na; beam diameter: 1 mm

9 Conventional vs VPP CTF behavior Conventional CTF - only the defocus varies - the CTF is pinned at k = 0 VPP CTF - both defocus and phase shift vary - the CTF is NOT pinned at k = 0 k = 0 k = 0

10 CTF fitting The CTF fitting is a very important step! The CTF fits provide valuable information about the technical quality of the dataset and the behavior of the phase plate.

11 CTF fitting improving the success rate Use proper search ranges for the defocus and phase shift. The ranges should match the actual ranges of those parameters in the data. Do not collect the data with too low of a defocus (<500 nm)! It is better to have a bit more defocus (~700 nm) than less. In order to improve the success rate of the fits we usually perform two rounds of CTF fitting. The first round with generic search ranges for the defocus and phase shift. The second round with search ranges corresponding to those measured in the first round. The high resolution limit should also be tuned according to the performance of the data?!? Always look at the diagnostic output files (*.ctf) to see what is wrong with the unsuccessful fits!

12 CTF fitting strategy Perform an initial fit with generic defocus (3000 to Å) and phase shift (10 to 150 deg) ranges Plot the measured defocus, phase shift and resolution as lines and histograms Determine the actual defocus and phase shift ranges Perform a second fit using the actual ranges as input parameters

13 (defu+defv)/2 CTF fitting defocus plots Defocus plot Defocus histogram Count micrograph Nr defocus [A]

14 phase shift [deg] Count CTF fitting phase shift plots Phase shift plot Phase shift histogram mincrograph Nr phase shift [deg]

15 resolution [A] CTF fitting resolution plots Resolution plot Resolution histogram Count mincrograph Nr resolution [A]

16 CTF fitting second fit Perform a second fit using the actual defocus and phase shift ranges measured from the first fit. Example Gctf command for the second fit: gctf --apix kv cs ac phase_shift_l 10 --phase_shift_h phase_shift_s 10 --phase_shift_t 1 -- dstep defl defh defs astm bfac resl resh boxsize do_epa 1 -- refine_after_epa 0 --convsize 30 --do_hres_ref 1 --Href_resL Href_resH Href_bfac 50 --estimate_b 1 --B_resL B_resH do_validation 1 micrographs/*.mrc

17 Micrograph resolution estimation be careful! NUCH1_050_May19_ ctf CC=0 (Gctf original) res. 2.3 A CC=0.75 res. 3.7 A CC<0 (GCTF) ~2.3 A

18 Micrograph resolution estimation be careful! NUCH1_050_May19_ ctf CC=0 (Gctf original) res. 2.3 A CC=0.75 res. 3.7 A CC<0.75 ~3.7 A CC<0 (GCTF) ~2.3 A

19 CTF fit resolution ranges NUCH1_349_May20_ ctf CC=0 (original) res A CC=0.75 res A CC<0.75 ~3.7 A CC<0 (GCTF) ~2.3 A

20 CTF fit resolution ranges NUCH1_349_May20_ ctf CC=0 (original) res A CC=0.75 res A initial fit range A CC<0.75 ~3.7 A CC<0 (GCTF) ~2.3 A

21 CTF fit resolution ranges NUCH1_349_May20_ ctf CC=0 (original) res A CC=0.75 res A high-res refinement A CC<0.75 ~3.7 A CC<0 (GCTF) ~2.3 A

22 abs(defu-defv) phase shift [deg] CTF fitting performance checks Astigmatism plot Phase shift plot micrograph Nr mincrograph Nr.

23 resolution [A] CTF fitting performance checks Resolution plot Resolution histogram Count mincrograph Nr resolution [A]

24 Frequency Hemoglobin dataset CTF fit statistics Fits with ctffind Estimated resolution cutoff Bin

25 Performance checkpoints Estimated CTF resolutions are a very good indicator of the technical quality of the dataset. The CTF resolutions histogram should be below 4 Å. Noise or spikes in the phase shift curve and/or the astigmatism curve indicate failed CTF fits. 2D classes represent the biochemical/cryo-em quality of the sample. Look for secondary structure elements a-helices. Determine the presence of flexible domains. Judge heterogeneity. Get an impression of the orientation distribution.

26 Use a phase shift prior?

27 CTF fitting in squared frequency space?

28 Effect of high phase shift on the CTF

29 Thank you for your attention!