DDDs, Screening micrographs and CTF(Practical work)

Vahid Abrishami

Introduction

• With direct detection devices (DDDs) low-dose images obtained by electron cryo-microscopy are recorded as frames.

• The DDDs have confirmed that the beam-induced motion (BIM) of the sample substantially degrades resolution.

• We need a correction for both global and local movements.

Import Movies

• Double-click the “import movies” box.

• Template has the right value for the acquisition parameters.

• Use the browse icon to select the path.

• Press the “Execute” button.

Import Movies

• A green box with finished status will appear.

• The summary tab should display some information such as the number of movies imported.

• We have a SetOfMovies.

Import Movies

• Press “Analyse Results” to check the list of movies.

Movie Alignment

• Aligning the individual frames of movies to correct the BIM image blurring and restore important high resolution information.

• The alignment method used in (Li et al., 2013) consists of a pure in-plane drift correction.

• The alignment method proposed in (Abrishami et al., 2015) is based on Optical Flow (OF) and works best at a local level.

Movie Alignment (Cont.)

• Open the “movie alignment” box.

• We can use the Search icon to select the SetOfMovies to be used as input.

• In this tutorial we use optical flow.

Movie Alignment (Cont.)

• First and Last frames in the alignment.

• In Advanced mode, removing the movies after alignment, and set the window size for OF can be asked.

Movie Alignment (Cont.)

• If there are GPUs, then we suggest to use first Li method and then OF.

Movie Alignment (Cont.)

• Click on “Analyse Results”.

Estimating CTF

• CTFs (Contrast Transfer Functions) of the micrographs can be estimated either using CTFFind (Mindell and Grigorieff, 2003) or Xmipp CTF estimation(Vargas, et.al, 2013 ).

• These protocols estimate the PSD (Power Spectral Density) of the micrographs and the parameters of the CTF (defocus U, defocus V, defocus angle, etc)

Estimating CTF (Cont.)

• Double click on the “ctffind4” box to edit.

• You can select ctffind3 or ctffind4 (the latter is reported to be ten times faster)

Estimating CTF (Cont.)

• Use the resolution wizard to set the low and high frequencies.

• All zeros of the PSD should be contained within those frequencies.

• Press “Execute”.

Estimating CTF (Cont.)

• Press “Analyse Results” to see a list of calculated CTF.

• Right click and select disable to reject a micrograph.

• Click Micrographs to have a new SetOfMicrographs.

Estimating CTF (Cont.)

• If rings are not properly estimated then right click and select Recalculate CTF.

• Finally press “Recalculate CTFs”.

Estimating CTF (Cont.)

• Analyze the CTF profiles by right-click on a micrograph row and selecting the “Show CTF profile” option.

Estimating CTF (Cont.)

• Right-click on a micrograph and select “Display CTF Fitting” to see the CTF fitting.

Estimating CTF (Cont.)

• To use XMIPP CTF, select XMIPP CTF from CTF estimation.

Estimating CTF (Cont.)

• This protocol can be used in the same way as ctffind4.

• There are some additional options in “Analyse Results”.

Estimating CTF (Cont.)

• You can check PSD, Envelope, and Background Noise (BGNoise).

References

• Abrishami, V., Vargas, J., Li, X., Cheng, Y., Marabini, R., Sorzano, C. O. S., , J. M., Carazo. "Alignment of direct detection device micrographs using a robust Optical Flow approach." Journal of structural biology 189.3 (2015): 163-176.

• C.O.S. Sorzano, S. Jonic, R. Núñez, N. Boisset, J.M. Carazo. Fast, robust and accurate determination of transmission electron microscopy contrast transfer function. Journal Structural Biology 160: 249-262 (2007).

• Li, Xueming, Paul Mooney, Shawn Zheng, Christopher R. Booth, Michael B. Braunfeld, Sander Gubbens, David A. Agard, and Yifan Cheng. "Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM." Nature methods 10, no. 6 (2013): 584-590.

• Vargas, J., Otón, J., Marabini, R., Jonic, S., de la Rosa-Trevín, J. M., Carazo, Sorzano, C. O. S. FASTDEF: Fast defocus and astigmatism estimation for high-throughput transmission electron microscopy. J. Structural Biology, 2013, 181, 136-148.