Materials and Methods
3.1 From nothing to high-resolution
3.1.3 Adding fake details to a structure
Protein complexes are not rigid objects. They have the capability to move from one confor-mation to another conforconfor-mation. These movements cause small changes in their structural representation (see section 1.1). Therefore, a refinement often fails to resolve highly dy-namic part at high resolution. In order to push the resolution within these dydy-namic parts, the classification with respect to these areas is done. The cryo-EM data is classified with respect to reference maps. As the previous experiment has shown that the alignment is
(a)Picked non single particles (b) Aligned non single
parti-cles (c)Re-projection of the
recon-structed map
Figure 3.9: Cryo-EM data from the overfitting noise experiment All six picked sections on the left side were identified on a single micrograph. These images matched to the reference projection. The middle images show the aligned, picked non-particles. These images were reconstructed to a 3D representation of the protein complex. The images on the right side are the re-projections of the reconstructed map.
vulnerable to model-bias, the following experiment demonstrates that the classification of cryo-EM data is also model-biased.
(a) Faked reference map (b) Reference map
Figure 3.10: Reference models for the classification Here, the two reference models used to classify cryo-EM data are shown. The gray structure on the left side is the ideal representation of the protein complex. The teal model to the right was modified. This model has the identical structure as the gray map with an additional density shaped like a skull. The enlarged section are identical regions with respect to the map. Here, it is stressed that the density model of the skull is not part of the original protein complex structure.
Design of experiment A synthetic atomic model representing the structure of a skull (enlarged image in Figure 3.10) was created by Dr. Niels Fischer2. WithChimera a density map of the fake atomic model was built. This density map was added to a high-resolution refined structure map of the 70S ribosome. A set of417,000single particle projection images with a box size of 420 and a pixel size of 0.75Å/pix were classified without additionally aligning to the two references of the ribosome in Figure 3.10 with RELION. 80% of the projection images were assigned to the original reference in Figure 3.10. The other 20%
showed a higher correlation to the faked-density reference in Figure 3.10. A gold-standard refinement was performed with the projection images which correlated better to the faked density. This computation was started using a reference structure that did not have the density of the skull to avoid reference biasing during alignment. It was possible because the data was already refined to an optimal structure and the imitation of the refinements was done with the known optimized parameter set. Important to notice is that there was no additional alignment procedure done. Here as well as before, the gold-standard refinement of the ribosome converged based on the correlation between the two maps.
Observation In Figure 3.11, the refined structure of one of the half maps and a meshed representation of the faked density model are shown. The refined 3D map of the ribo-some contains a coarse density that fits to the skull reference. The enhanced side view in Figure 3.11b has a visible reconstructed density, where the atomic model of the skull was appended to the ribosome reference in Figure 3.10a. The top view of the map in Figure 3.11a, further, shows visible features of the silhouette of the faked density. The reconstructed density and the modeled density visually differ in their feature resolution.
This, indeed, helps the reliability of the refined structure. To emphasize again, the skull is a faked atomic model, which was added to the ribosome map. The cryo-EM data was classified to this faked reference so that it was forced to detect a variation of the faked skull density in the recorded cryo-EM data. With the knowledge that the single particle projection images do not contain a signal similar to the skull structure, the classification fitted noise into the variation of the faked projected density. However, the classified cryo-EM was gold standard refined without the faked density reference in Figure 3.10a. The refinement should be free of the model bias based on the faked reference. Knowing the ribosome structure from previous experiments and the fake reference, the refined density of the skull is a contribution of the classified noise within the original single particle images.
If the perfect density of the skull would have been reconstructed, it could be more obvious that there is something falsely detected in the cryo-EM data.
Both half maps have a representation of the reconstructed skull density such that the FSC in Figure 3.12 estimates a resolution of around4.1Å for the reconstruction with faked
2Department of Structural Dynamics, Max Planck Institute for Biophysical Chemistry
(a) Top view (b) Side view
Figure 3.11: Refined structure of model-biased classified dataThe teal map in this figure is the reconstruction of the classified data with RELION. The two enlarged regions of the 3D reconstructed map (teal) show the recovered faked density in more detail. The meshed structure shaped as skull denotes a part of reference density, which was used to classify the data. The right image shows the top view of the reconstruction. The left image section shows a side-view of the 3D map.
density classified data (resp. 6.3 Å for the conservative threshold). Moreover, the FSC shows the general characteristics of the correlation curve between reconstructed proteins.
Consequently, the FSC does not detect the mistakenly reconstructed noise and its resulting quality issues of the ribosome density. The estimated resolution of 4.1 Å is worse than the2.9Å published structure of the protein complex recovered from the identical cryo-EM data set [7]. One reason is the number of refined particles. Instead of refining 417k single particles only 83k projection images were reconstructed. Additionally, the resolution of the published map was estimated with the FSC between two auto-masked half maps. Using the auto-masking option in RELION the FSC for the experiment in Figure 3.12 determines a resolution of 3.1 Å (resp. conservative threshold 3.9 Å). The difference of 0.2Å is most likely a consequence of the number of projection images used to reconstruct.
Figure 3.12: FSC of model-biased classified data Here, the unmasked and masked FSCs between the two half-set maps are plotted. The refinement was done with approximately 20% of the raw data, which classified well to the faked density reference map (the teal map in Figure 3.10).