Structural Biology

Biography

Biography: Thomas Braun

Abstract

Cryo-electron microscopy (cryo-EM) sample preparation techniques ensure that biological specimens can be investigated at physiological conditions in the electron microscope.  However, these preparation methods suffer from extensive blotting steps leading to a massive loss of sample and sometimes to partial denaturation of sensitive protein complexes. We have developed a simple method for the almost lossless conditioning and preparation of nanoliter-volumes of biological samples for EM. The method does not involve any blotting steps. A microcapillary is used to aspirate 3 to 20 nanoliters of sample, depending on the experiment (Figure 1A). The sample is applied (left) and spread (center) on the EM-grid. Real-time monitoring allows the thickness of the water film to be assessed and decreased to the optimum value prior to vitrification (right). We prepared cryo-EM grids of various samples, e.g., bacteriophages and soluble proteins as shown in Figure 1B&C, to demonstrate the usefulness and general applicability of the method. We also showed that high-resolution 3D structures can be calculated from single-particle preparations of a soluble protein. In addition to cryo-EM grid preparation, the versatile method allows nanoliter-sized sample volumes to be conditioned for EM, e.g., negatively stained with heavy metal salts or embedded in trehalose.

In addition, we combine the new sample preparation method with a single cell lysis device for adherent eukaryotic cells and image the aspirated cell contents by TEM. To demonstrate the usefulness of this new “visual proteomics” approach we visualized the changes occurring in single cell proteomes upon heat shocking the cells. Furthermore, we have developed a protein-fishing method based on a magnetic trap and photo-cleavable composite material, to ‘fish’ untagged proteins from cell lysate by antibodies. This allows target proteins to be isolated from approx. 40’000 cells in 90 min and analysed by EM. 

References:

1. Kemmerling S, Ziegler J, Schweighauser G, Arnold S A, Giss D, Müller S A, Ringler P, Goldie K N, Goedecke N, Hierlemann A, Stahlberg A, Engel A, Braun T (2012) Connecting µ-fluidics to electron microscopy. Journal of Structural Biology, 177:1, 128–134.
2. Arnold S A, Albiez S, Opara N, Chami M, Schmidli C, Bieri A, Padeste C, Stahlberg H, Braun T (2016) Total sample conditioning and preparation of nanoliter volumes for electron microscopy. ACS Nano, 10:  5, 4981–4988.
3. Arnold S A, Albiez S, Bieri A, Syntychaki, A, Adaixo R, McLeod R A, Goldie K N, Stahlberg H, Braun T. (2016) Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts. Journal of Structural Biology, in press.
4.  Kemmerling S, Arnold S A , Bircher B, Sauter N, Escobedo C, Dernick G, Hierlemann A, Stahlberg H, Braun T (2013) Single-cell lysis for visual analysis by electron microscopy. Journal of Structural Biology, 183:  3, 467–473.
5.  Giss D, Kemmerling S, Dandey V, Stahlberg H, Braun T (2014) Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy. Analytical Chemistry, 86: 10, 4680–4687.