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Fast protein-complex isolation, sample preparation and data processing for high-resolution structural analysis and visual proteomics

Applicant Braun Thomas
Number 162521
Funding scheme Project funding (Div. I-III)
Research institution C-CINA Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Other disciplines of Physics
Start/End 01.02.2016 - 30.04.2021
Approved amount 558'944.00
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All Disciplines (2)

Discipline
Other disciplines of Physics
Biophysics

Keywords (5)

Biophysics; Structural biology; Microfluidics; Protein dynamics; Electron microscopy

Lay Summary (German)

Lead
Neue Präparationsmethoden für hochauflösende Elektronenmikroskopie
Lay summary

In den letzten Jahren hat die Elktronenmikroskopie eine veritable Renaissance erlebt. Drei technische Errungenschaften haben diese Entwicklung ermöglicht: Erstens wurden Probenpräperationemthoden entwickelt, die biologisches Material unter physiologischen Bedingungen so stabilisieren, dass sie für die Elektronenmikroskopie geeignet sind - die sogenannte "cryo Elektronenmikroskopie". Zweitens werden neue Elektronenquellen eingesetzt, die eine bessere Beleuchtung der Proben im Mikroskope erlauben. Drittens wurden in den letzten drei Jahren neuartige Kameras entwickelt, sogenannte "direct electron detectors", die Bilder in ungeahnter Qualität aufnehmen können. Diese drei Errungenschaften, zusammen mit verbesserten Computeralgorithemen, erlauben es heute Biomoleküle, zum Beispiel Proteinkomplexe, atomar aufzulösen, ohne dass diese zuvor in einem Kristall angeordnet werden müssen.

 Im Gegensatz dazu haben sich die Reinigungsverfahren die nötig sind, um bestimmte Bestandteile einer biologischen Zelle, wie etwa Proteinkomplexe, zu isolieren, die letzten 20 Jahre kaum verändert: Es sind immer noch grosse Mengen an Ausgangsmaterial notwendig, und die Reinigungsverfahren sind langwierig. Diese Projekt hat sich zum Ziel gesetzt, diesen Flaschenhals zu beseitigen und neue Verfahren mittels mirkofluidischen Methoden zu entwickeln, um aus wenig Ausgangsmaterial und in kürzester Zeit Biomoleküle zu isolieren und direkt für die Elektronenmikrokopie aufzubereiten.

Wir versprechen uns von solchen neuen Verfahren weitere Fortschritte in der biologischen und biomedizinischen Grundlagenforschung, vor allem was die strukturelle Untersuchung von grossen und empfindlichen Proteinkomplexen betrifft. 

Direct link to Lay Summary Last update: 04.01.2016

Responsible applicant and co-applicants

Employees

Project partner

Publications

Publication
Microfluidic protein isolation and sample preparation for high-resolution cryo-EM
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.
“Differential Visual Proteomics”: Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells
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.
Miniaturized Sample Preparation for Transmission Electron Microscopy
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.
Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”
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.
Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts
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.
Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.
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.

Datasets

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).

Endogeneous native human 20S proteasome with bound Fabs isolated from less than 1 ul cell lysate

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.

Cryo-EM structure of Tobacco Mossaic Virus from microfluidic grid preparation

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).

Tobacco Mosaic 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)

Collaboration

Group / person Country
Types of collaboration
Prof. Marcel Leist, Konstanz Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Andreas Hierlemann, BSSE, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Timm Meier, Biozentrum, Universität Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
2nd Cryo-EM User Symposium at EMBL Heidelberg Talk given at a conference The cryoWriter: Modular microfluidics for protein isolation, EM-grid preparation, and 'differential visual proteomics.' 07.02.2020 Heidelberg, Germany Braun Thomas;
3rd Instruct Workshop for Best Practices in CryoEM Talk given at a conference The cryoWriter: Modular microfluidics for protein isolation, EM-grid preparation, and 'differential visual proteomics.' 19.11.2019 Strasbourg, France Rima Luca;
nternational Symposium onStructure Biology for Drug Discovery@SwissFEL Talk given at a conference icrofluidic Sample Preparation for EM: Protein Isolation,High-Resolution cryo-EM, and 'Differential Visual Proteomics’ 25.06.2019 Villigen (PSI), Switzerland Braun Thomas;
CCP-EM Spring Symposium 2019 Talk given at a conference Mircrofluidic protein preparation and cryo-EM sample preparation for high resolution structure determination 29.03.2019 Nottingham, Great Britain and Northern Ireland Braun Thomas;
Structural Dynamics in Cellular Communication (2nd edition) - BECM Sattelite meeting Talk given at a conference Microfluidic EM sample preparation for differential visual proteomics and diagnostics 19.09.2018 Brussels, Belgium, Belgium Braun Thomas;
Thermo Fisher Scientific Cryo-EM User Group Meeting 2018 Talk given at a conference Microfluidic sample preparation for electron microscopy 24.04.2018 eindhoven, Netherlands Braun Thomas;
3rd International Symposium on Cryo-EM 3D Image Analysis Poster A single-particle EM approach for differential visual proteomics 21.03.2018 Lake Tahoe, California, USA, United States of America Stahlberg Henning; Syntychaki Anastasia;
NRAMM 2017: A Workshop on Advanced Topics in EM Structure Determination: Challenges and Opportunities Talk given at a conference Microfluidic Sample Preparation: Opportunities, Challenges and ‘Visual Proteomics’ 29.10.2017 New York, United States of America Braun Thomas;
Gordon research conference: Three Dimensional Electron Microscopy Talk given at a conference Microfluidic Sample Preparation for Electron Microscopy: Opportunities, Challenges and 'Visual Proteomics 11.06.2017 Diablerets, Switzerland Braun Thomas;


Use-inspired outputs


Start-ups

Name Year
cryoWrite 2020

Associated projects

Number Title Start Funding scheme
192190 Microfluidic Sample Preparation for High-Resolution Electron Microscopy, Visual Proteomics and Electron Tomography 01.03.2021 Project funding (Div. I-III)

Abstract

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”).
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