1. CTF sensitivity • How do the errors in the estimation of the CTF affect the CTF shap itself and the final reconstruction? • Which are the most important CTF parameters? • How do the errors in one parameter affect other parameters?

2. Which CTF? 1D CTF 1D PSD 2D PSD and CTF

3. Which CTF?

4. Which CTF?

5. How to measure the effect of errors? What happens to the ideal CTF if I change a little bit my current estimate of the CTF parameters? What if I change a little bit only one parameter? Can you express this as percentages? Sensitivity=How sensitive is the CTF to the changes in this parameter What is the average sensitivity?

6. So, which is the effect of each parameter on the CTF, PSD and 1st zero of the CTF? The most important for the CTF: Microscope voltage, defoci, and chromatic aberration The most important for the PSD: Background parameters, microscope voltage and defoci The most important for the first zero: Microscope voltage, defoci, and fraction of scattered electrons

7. <0.01% JEOL Which is the accuracy in the determination of the important parameters? These accuracies have been estimated by bootstrapping on the PSD values of a micrograph and reestimating for each subsample the CTF.

8. Do these errors affect the final reconstruction? In our experiments, it did not significantly affect the phase correction

9. Do these errors affect the final reconstruction? Neither the amplitude correction

10. Can I remove the insensitive parameters? Only the drift parameters, since in fact in these micrographs, there is no drift!! The rest are insensitive (you can commit relatively large errors), but that does not mean you can remove them. If you remove them, then very bad CTFs are estimated

11. If I commit an error estimating one parameter, how this affects the rest? There are groups of parameters and changes in one parameter can be compensated by others. For instance, in the following table we see that changes in the major defocus are negatively correlated to changes in the scattered fraction and positively correlated to changes in the minor defocus. These results have been achieved through the bootstrapping strayegy already described.

12. If I commit an error estimating one parameter, how this affects the rest? This is to difficult for me to understand, can you further clarify who affects who? Of course, through factor analysis we can discover groups of parameters strongly influencing each other General background Background medium freq. Ideal CTF Background low freq. CTF decay due to incoherence Background low freq. CTF decay due to energy spread