Negative-stain EM of yeast tri-snRNPs

We gathered sufficient biochemical evidence, which compelled us to modify the typical standard yeast tri-snRNP protein profile. The tri-snRNP now contains stably associated Sad1 and Prp28, while the amounts of Prp38 remain comparatively low. Also the fact that the yeast tri-snRNP is now stable under ATP conditions makes the yeast particle more similar to its human counterpart. Therefore it was interesting to see whether the Brr2 would assume a conformation in our Sad1-TAP tri-snRNP like observed for Brr2 in the human tri-snRNP.

We therefore set out to investigate the structure of the yeast tri-snRNP purified via Sad1-Tag.

We started preliminary structure investigation with negative-stained EM analysis. Tri-snRNP particles were accordingly purified, fixed, and negatively stained using a well-established protocol for negative EM analysis. Fixed purified particles were adsorbed on EM grids, stained for suitable duration, and later inspected under a CM 200 electron microscope. In collaboration with my colleagues, we performed EM analysis, and collected negative stain-EM micrographs of Sad1-TAP tri-snRNP particles. Micrographs collected from the negative stain-EM were subjected to software-based as well as manual particle selection. The refined data set enabled us to obtain a number of 2D class-averages of the Sad1-Tap tri-snRNP. The entire dataset generated close to 250 different 2D class-averages with each class consisting of particles with a similar view.

The 2D classes from negative-stained micrographs displayed structural details of the triangular particles with high contrast. Visible is a main 'body' with pointed lower end and a broad 'head'

structure along with a small 'arm' like structure connected to the main central body by a tiny linker region (fig: 3.17, B). The most prominent classes showed similar structural features that resemble an overall view of the low resolution tri-snRNP model, which had already been reported by our laboratory (Häcker et al., 2008). The two major class-averages showed two different prominent views: one with the 'closed' conformation, and the other with a more 'open' conformation. 'Open' and 'closed' conformation relates to a different positioning of the head and the arm domains relative to each other on a fixed central main body (fig: 3.17, C and D). These results are consistent with the previous findings supporting the dynamics of tri-snRNP particles with respect to the highly flexible 'head' domain.

Later we compared the 2D averages of the yeast SAD1-TAP tri-snRNP with the 2D class-averages obtained from negative EM studies of the human tri-snRNP (human negative stained EM 2D classes were kindly provided by Cole Townsend, Department for Structural Dynamics, MPI-BPC).

Figure 4.1 Comparison of 2D class-averages of Sad1-TAP purified yeast tri-snRNPs with human tri-snRNP 2D class-averages. Upper panel; shown here are various predominant views (2D classes) obtained from the negative-stain EM 2D data analysis of human tri-snRNP particles. With three different views for somewhat similar and slightly rotated views in upper panel box-1, and in box 2 and 3, other prominent views in the negative-stain EM human tri-snRNP data set. Lower panel: shown here are the selected final representative 2D class averages of yeast Sad1-TAP tri-snRNP particles that may bear similar prominent features as observed in the case of human tri-snRNPs. Lower panel box 4 contains three different views that are potentially in a similar orientation as shown for the human tri-snRNP in the upper panel box 1. Lower panel box 5 and box 6 also show striking resemblance to representative views of the human tri-snRNPs in box 2 and box 6 respectively (human tri-snRNP 2D class average pics were kindly provided by Cole Townsend - Department of Structural Dynamics, MPI-BPC)

Interestingly, 2D comparison revealed the existence of particles that populate some subclasses with few members only. These subclasses harbour particles with compact topographies, which resemble the typical features of the most prominent class-averages of the negatively-stained human tri-snRNPs particles (see fig: 4.1.). One has to keep in mind though, that 2D similarities do not permit any conclusions related to possible 3D similarities. The predominant views in the class-averages of Sad1-TAP tri-snRNP particles show an overall view similar to the reported yeast tri-snRNP particles. These observations, with all due caution, could possibly be interpreted as an equilibrium between a dominant yeast-like conformation of the tri-snRNP and a minor population of human-like tri-snRNPs. Support for such a scenario stems from crosslinking data collected for the Sad1-TAP tri-snRNP and described in sections 3.3.1. and 3.3.2. Not only were these crosslinks helpful in determining the location of Sad1, but certain crosslinks and crosslinking distances were clearly only compatible with a sub-population of yeast tri-snRNPs in an assumed human-like conformation. These crosslinks made it even possible to design in silico a

"human-like" yeast tri-snRNP.

Using the same negative-stain EM dataset, we used the 3D electron density map of the Sad1-TAP tri-snRNP to interpret structural features with more confidence. The dataset resulted in the reconstruction of a 3D model very similar to the published 3D structures of the yeast tri-snRNP.

It was therefore even more interesting to pin-point the location of factors (such as Sad1 and Prp28) which are exclusively associated with our Sad1-TAP tri-snRNP purified according to the new protocol. The 3D model reconstruction with negatively-stained EM particles had the disadvantage that we could only observe the overall features. In order to understand the location and orientation of certain protein factors which had never been annotated in published yeast tri-snRNP models before, we needed to further analyse the Sad1-TAP tri-tri-snRNPs by cryo-EM with hoping to get a higher resolution 3D model.

The 3D model derived from negative-stained EM of Sad1-TAP tri-snRNPs, again confirmed that Sad1-TAP tri-snRNP particles have a Brr2 in a similar orientation as already shown in the published yeast tri-snRNP model. However, we could not reconstruct a 3D model using the limited number of particles in minor sub-classes. (particles with unique human-like orientation).