Biophysics; Structural biology; Microfluidics; Protein dynamics; Electron microscopy
Schmidli Claudio, Albiez Stefan, Rima Luca, Righetto Ricardo, Mohammed Inayatulla, Oliva Paolo, Kovacik Lubomir, Stahlberg Henning, Braun Thomas (2019), Microfluidic protein isolation and sample preparation for high-resolution cryo-EM, in
Proceedings of the National Academy of Sciences, 201907214-201907214.
Syntychaki Anastasia, Rima Luca, Schmidli Claudio, Stohler Thomas, Bieri Andrej, Sütterlin Rosmarie, Stahlberg Henning, Castaño-Díez Daniel, Braun Thomas (2019), “Differential Visual Proteomics”: Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells, in
Journal of Proteome Research, 18(9), 3521-3531.
Schmidli Claudio, Rima Luca, Arnold Stefan A., Stohler Thomas, Syntychaki Anastasia, Bieri Andrej, Albiez Stefan, Goldie Kenneth N., Chami Mohamed, Stahlberg Henning, Braun Thomas (2018), Miniaturized Sample Preparation for Transmission Electron Microscopy, in
Journal of Visualized Experiments, (137), 1-12.
Arnold Stefan A., Müller Shirley A., Schmidli Claudio, Syntychaki Anastasia, Rima Luca, Chami Mohamed, Stahlberg Henning, Goldie Kenneth N., Braun Thomas (2018), Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”, in
PROTEOMICS, 18(5-6), 1700176-1700176.
Arnold Stefan A., Albiez Stefan, Bieri Andrej, Syntychaki Anastasia, Adaixo Ricardo, McLeod Robert A., Goldie Kenneth N., Stahlberg Henning, Braun Thomas (2017), Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts, in
Journal of Structural Biology, 197(3), 220-226.
Arnold Stefan A, Albiez Stefan, Opara Nadia, Chami Mohamed, Schmidli Claudio, Bieri Andrej, Padeste Celestino, Stahlberg Henning, Braun Thomas (2016), Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy., in
ACS nano, 10(5), 4981-8.
CryoWriter: 3.5 Å structure of human 20S proteasome with bound Fabs from microfluidic protein isolation, and 1.9 Å TMV structure
| Author |
Schmidli, Claudio; Albiez, Stefan; Righetto, Rocardo; Mohammed, Inay; Oliva, Paolo; Kovacik, Lubomir; Stahlberg, Henning; Braun, Thomas |
| Publication date |
21.02.2019 |
| Persistent Identifier (PID) |
EMD-4738 |
| Repository |
Electron Microscopy Public Image Archive (EMPIAR)
|
| Abstract |
Micrographs of cryo-EM sample prepared using a microfluidic portein extraction and preparation system (cryoWriter).
| Author |
Schmidli, Claudio; Albiez, Srafan; Rima, Luca; Righetto, Ricardo; Mohammed, Inay; Oliva, Paolo; Kovacik, Lubomir; Stahlberg, Henning; Braun, Thomas |
| Publication date |
03.07.2019 |
| Persistent Identifier (PID) |
http://europepmc.org/abstract/MED/31292253 |
| Repository |
Protein Data Bank in Europe
|
| Abstract |
single particle analysis. Final map at 3.5 A overall resolution. Containes all 14 protein units of the core complex and, attached, two Fab fragments against the alph-4 subunit.
| Author |
Schmidli, Claudio; Albiez, Stafan; Rima, Luca; Righetto, Riccardo; Mohammed, Inay; Oliva, Paolo; Kovacik, Lubomir; Stahlberg, Henning; Braun, Thomas |
| Publication date |
03.07.2019 |
| Persistent Identifier (PID) |
http://europepmc.org/abstract/MED/31292253 |
| Repository |
Protein Data Bank in Europe
|
| Abstract |
Resolution control of microfluidic sample preparation. 1.9 A map of tobacco mosiac virus (TMV).
| Author |
Schmidli, Claudio; Albiez, Stafan; Rima, Luca; Righet to, Riccardo; Mohammed, Inay; Oliva, Paolo; Kovacik, Lubomir; Stahlberg, Henning |
| Publication date |
29.03.2019 |
| Persistent Identifier (PID) |
http://doi.org/10.2210/pdb6R7M/pdb |
| Repository |
Protein Data Bak (PDB)
|
| Abstract |
Structure of the Tobacco Mosaic Virus. Based on the 1.9 A structure (https://www.emdataresource.org/EMD-4628).
Endogeneous native human 20S proteasome
| Author |
Schmidli, Claudio; Albiez, Stafan; Rima, Luca; Eighetto, Riccardo; Mohammed, Inay; Oliva, Paolo; Kovacik, Lubomir; Stahlberg, Henning |
| Publication date |
03.07.2019 |
| Persistent Identifier (PID) |
6R70 |
| Repository |
Protein Data Bank (PDB)
|
| Abstract |
Atomic model of all 14 subunits of the human proteasome 20S core. Based on the 3.5 A structure (https://www.emdataresource.org/EMD-4738)
Direct electron detection (DED) cameras for electron microscopes introduced a fast and lasting change to biophysics and structural biology. These cameras now allow the structure determination of large biomolecules by cryo-electron microscopy (cryo-EM) at or close to atomic resolution using a single particle approach, without crystallization. However, protein isolation techniques and sample preparation methods for cryo-EM remain a bottleneck. Hence, advanced methods for protein isolation and sample preparation for high-resolution electron microscopy are urgently needed, especially when large (and unstable) protein assemblies are targeted.New methods must overcome several hurdles: (i) the protein complexes must be produced in significant amounts for subsequent structural analysis. Unfortunately, many protein complexes of (biomedical) interest are sparsely produced in eukaryotic cells. (ii) The destabilization of complexes during isolation must be countered. Protein assemblies are significantly diluted on isolation and their inter-molecular interactions are destabilized. This leads to the dissociation of many complexes formed transiently during biological processes. (iii) The data analysis of heterogeneous samples, as they arise due to the stochastic interaction networks, is still cumbersome. Nevertheless, precisely these “interactomes” are of great interest in biological research.The above difficulties can be avoided by, first, minimizing the overall sample consumption, and, second, by reducing the time required to isolate the target complexes and prepare samples for cryo-EM. Protein isolation should be fast (one step, approx. 1h) and produce samples clean enough for imaging by cryo-EM. Ideally, the protocol should be independent of protein modifications, such as tags, as these can interfere with the biological function and assembly of the target complex. Additionally, stabilization of the complex by new cross-linking methods might be desirable directly after cell lysis. Last but not least, sample preparation for cryo-EM should be accomplished in a loss-less manner: to date, more than 99% of the protein is lost during blotting steps when classical cryo-EM grid preparation methods are used.This project aims (i) to establish a method to rapidly extract target proteins and their complexes from minimal amounts of cell lysate, and, (ii) to develop a loss-less cryo-EM grid preparation system that only consumes minute amounts of sample (5 nl) and does not involve any blotting steps. In this framework we will also explore the use of microfluidic cross-linking strategies to stabilize protein-complexes. In addition, methods for structural analysis by the single particle cryo-EM approach and de novo identification of complex subunits or interaction partners will be developed and tested on a single particle level (“visual proteomics”).