Blotting protein complexes from native gels to electron microscopy grids

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1 Nature Methods Blotting protein complexes from native gels to electron microscopy grids Roland Wilhelm Knispel, Christine Kofler, Marius Boicu, Wolfgang Baumeister & Stephan Nickell Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Table 1 Native gel of crude cytosolic extract Reference projections of the analyzed complexes Electron micrographs of 26S proteasomes blotted from native gels Integration of grid blotting in automated protein complex analysis Mass spectrometric identification of protein bands after applying the grid blotting procedure

2 Supplementary Figure 1: Native gel of crude cytosolic extract An amount of a freshly prepared cytosolic extract of Thermoplasma acidophilum, equalling to 40 µg of protein, was loaded next to a molecular weight marker on a native 3-8 % TA gel and visualized by Coomassie staining after electrophoresis (a). The gel demonstrates the complexity of crude cell protein extracts, which has to be reduced by using fractionation techniques, to make investigations of individual proteins feasible.

3 Supplementary Figure 2: Reference projections of the analyzed complexes The displayed projections were generated by converting PDB files of Thermosome (PDB accession code: 1PMA) and 20S proteasome (1A6D) to EM maps, followed by low-pass filtering (a,b) or by low-pass filtering the previously published EM density of VAT.

4 Supplementary Figure 3: Electron micrographs of 26S proteasomes blotted from native gels Purified 26S proteasomes from Drosophila melanogaster were subjected to native gel electrophoresis on 3-8 % TA gels (a). Visualized bands reveal various assembly stages of the holocomplex, ranging from fully assembled 26S proteasomes (top band, 2.5 MDa), to 20S proteasomes associated with only one regulatory particle or with partially assembled regulatory particles. The lower band shows single 20S proteasomes. This has been confirmed by mass spectrometric analysis. After electrophoresis of 5 and 10 µg purified protein, the upper band was blotted and prepared by negative staining (b,c), respectively. We observed an increase of protein aggregates with higher loading amounts (10 µg), although the number of single particles observed remained approximately constant. The applied defocus setting was - 3 µm. Scale bars correspond to 100 nm.

5 Supplementary Figure 4: Integration of grid blotting in automated protein complex analysis Protein mixtures are pre-fractionated by a suitable method to reduce sample complexity prior to native gel electrophoresis. After electrophoretic separation small amounts of protein complexes, focussed in bands, are transferred to an electron microscopy grid by means of the grid blotting technique. Proteins are negatively stained or ice-embedded prior to single particle electron microscopy. Subsequently, a gel sample from the same protein band is excised for mass spectrometric analysis. To facilitate an automated grid blotting procedure, the protocol described in this communication has to be further improved. Ideally, an imaging technique for the direct detection of protein bands without the need of duplicate samples and gel staining would be implemented. A viable approach would be a protein detection method based on intrinsic fluorescence activity of aromatic amino acid residues, when excited with far UV light 13 ( nm). Here, care has to be taken to avoid radiation damage and crosslinking of the gel, calling for low dose illumination schemes. The digital fluorescence

6 image could be used to guide a robotic arm to the protein bands, where the blotting and the preparation of the EM grids are carried out in an automated fashion. Such a blotting mechanism could be combined with a spot-picker setup suitable for MS sample preparation. This way, a large number of protein bands could be processed and analyzed in parallel, thus creating a platform for comprehensive proteome-wide screening of protein complexes in conjunction with high-resolution structure determination (filed patent application PCT/EP2005/004600: Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.v.).

7 Supplementary Table 1: Mass spectrometric identification of protein bands after applying the grid blotting procedure SwissProt Accession Number sp P25156 PSMA_THEAC sp P28061 PSMB_THEAC Protein Description Proteasome subunit alpha Proteasome subunit beta Score Mass / Da Unambiguous Matches Sequence Coverage / %