Figure 3.
Refinements with Differing Parameters to Test the 3D CTF Model and to Probe the Limits of the Dataset
(A) Using conventional strip-based CTF correction implemented in IMOD, automatic refinement in RELION yielded a 10.9 Å resolution map. Only phase flipping and no amplitude weighting was conducted in this CTF correction.
(B) Compared with the refinement using our new 3D CTF model, the resolution obtained was lower.
(C) Resolution of sub-tomogram averaging reconstruction could not be improved by adding more data or removing subsets of the data. Different sub-tomogram averaging refinements were conducted using random subsets of the data of different sizes. A similar resolution was obtained with fewer particles, showing that the refinement was not limited by the size of the dataset.
(D) Part of the data that had been exposed to a cumulative electron dose of > 20 or 30 e−/Å2 was removed using the 3D CTF model. Compared with the full dataset in which 60 e−/Å2 had been applied to the specimen, no improvement was observed (the weighted 3D CTF model was used in all refinements).
(E) Reference-free refinement using the same data as shown in Figure 2. Scale same as (A).
(F) The resolution of the output, refined structure (11.5 Å) was not as high as in cases when a reference was used (9.2 Å).