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High-Resolution Membrane Protein Structures by Cryo-EM

Applicant Stahlberg Henning
Number 166164
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 01.04.2016 - 31.03.2019
Approved amount 540'000.00
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All Disciplines (2)

Discipline
Other disciplines of Physics
Biophysics

Keywords (9)

maximum likelihood; software; image processing; 2dx; electron crystallography; structure; hybrid input output algorithm; cryo-electron microscopy; membrane protein

Lay Summary (German)

Lead
In diesem Projekt werden wir Computer Bildverarbeitungs-Software erstellen, welche es erlaubt, aus Elektronenmikroskopie Bildern von dicht gepackten Membranproteinen die Struktur der Proteine zu ermitteln.
Lay summary

Membranproteine sind biologische Nanomaschinen, welche in den Membranen unserer Zellen sitzen, und dort wichtige biologische Funktionen ausführen. Membranproteine können zum Beispiel Nahrungsstoffe in die Zellen hindurch lassen oder in die Zellen pumpen, oder sie können Abfallprodukte aus den Zellen heraus pumpen. Manche Membranproteine können auch als Sensoren dienen, wenn sie zum Beispiel kleinste Mengen von Molekülen detektieren, wie zum Beispiel ein Hormon oder Salz, um anschliessend mechanisch darauf zu reagieren.

Für die medizinische Forschung ist es wichtig, die Struktur der Membranproteine zu kennen. Denn mit der Struktur kann die Funktion der Membranproteine verstanden werden, was hilfreich ist, wenn ein Medikament entwickelt werden soll, welches ein bestimmtes Membranprotein in seiner Funktion unterstützen oder blockieren soll.

Die Struktur von Membranproteinen kann klassischerweise mit einem Elektronenmikroskop ermittelt werden, wenn das Membranprotein in eine biologische Membran eingebaut wird, und dort in regelmässigen Positionen arrangiert wird. Solche sogenannten 2D Kristalle können dann mit dem Elektronenmikroskop abgebildet werden, und Computer Bildverarbeitung erlaubt dann, aus solchen Bildern die detaillierte Struktur der Proteine zu errechnen.

In diesem Projekt werden wir unsere bestehende Bildverarbeitungs-Software "2dx" um neue Algorithmen erweitern, mit dem Ziel, die Auflösung der Proteinstrukturen zu verbessern, die Geschwindigkeit der Strukturbestimmung zu beschleunigen, und, was am wichtigsten ist, um die Notwendigkeit der regelmässigen Anordnung der Membranproteine zu eliminieren. Wenn unsere Software in der Lage ist, aus Bildern von nur dicht gepackten aber nicht 2D kristallisierten Proteinen die Struktur zu bestimmen, wäre dies ein sehr grosser Gewinn für die Strukturbestimmung, da der mühsame Prozess der Membranprotein-Kristallisation entfallen würde. 

Direct link to Lay Summary Last update: 04.01.2017

Responsible applicant and co-applicants

Employees

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, 116(30), 15007-15012.
Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM
Righetto Ricardo D., Biyani Nikhil, Kowal Julia, Chami Mohamed, Stahlberg Henning (2019), Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM, in Nature Communications, 10(1), 1722.
High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer.
Kowal Julia, Biyani Nikhil, Chami Mohamed, Scherer Sebastian, Rzepiela Andrzej J, Baumgartner Paul, Upadhyay Vikrant, Nimigean Crina M, Stahlberg Henning (2018), High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer., in Structure (London, England : 1993), 26(1), 20-27.
Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I
Bibow Stefan, Polyhach Yevhen, Eichmann Cédric, Chi Celestine N, Kowal Julia, Albiez Stefan, McLeod Robert A, Stahlberg Henning, Jeschke Gunnar, Güntert Peter, Riek Roland (2017), Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I, in Nature Structural & Molecular Biology, 24(2), 187-193.
Focus: The interface between data collection and data processing in cryo-EM.
Biyani N, Righetto RD, McLeod R, Caujolle-Bert D, Castano-Diez D, Goldie KN, Stahlberg H (2017), Focus: The interface between data collection and data processing in cryo-EM., in Journal of Structural Biology, 198(2), 124-133.
Image processing techniques for high-resolution structure determination from badly ordered 2D crystals
Biyani Nikhil, Scherer Sebastian, Righetto Ricardo D., Kowal Julia, Chami Mohamed, Stahlberg Henning (2017), Image processing techniques for high-resolution structure determination from badly ordered 2D crystals, in Journal of Structural Biology, 203(2), 120-134.
Robust image alignment for cryogenic transmission electron microscopy.
McLeod RA, Kowal J, Ringler P, Stahlberg H (2017), Robust image alignment for cryogenic transmission electron microscopy., in Journal of Structural Biology, 197(3), 279-293.

Scientific events



Self-organised

Title Date Place
Single Particle Cryo-EM Workshop 2018 at C-CINA 22.08.2018 Uni Basel, Switzerland
2DX Workshop 2016 22.08.2016 University of Basel, Switzerland

Associated projects

Number Title Start Funding scheme
144427 Software for Electron Microscopy of Membrane Proteins 01.10.2012 Project funding (Div. I-III)
177084 Efficient cryo electron microscopy of macromolecular assemblies and membrane proteins 01.09.2018 R'EQUIP

Abstract

Membrane proteins are central to health and disease. Membrane proteins thereby function within the lipid membranes of cells, where they are responsible for a plethora of functions. Among others, they facilitate or regulate transport of molecules across the membrane, or sense ligands, membrane potential, or buffer gra-dients to react. Cryo-transmission electron microscopy (cryo-EM) classically allows studying the high-resolution structure of membrane proteins, if these are lipid membrane-reconstituted and two-dimensionally crystallized. This method is called electron crystallography of membrane proteins. My group has developed an image processing package for the analysis of 2D crystals, called the 2dx software package. Cryo-EM of isolated single particles has seen a major breakthrough recently, through the introduction of di-rect electron detector (DED) cameras and the development of improved single particle image processing algorithms. Single-particle cryo-EM allows to rapidly determine the atomic resolution structure of detergent-solubilized membrane proteins without the need for crystallization. This, however, does not allow the direct study of the structure of membrane proteins in a full lipid membrane, as electron crystallography provides. In this project, we will extend the 2dx software suite to accelerate processing, to automate processing, to increase resolution of the 3D reconstructions, and to make this method largely independent of highly ordered 2D crystals, so that also membrane-reconstituted membrane proteins that are not - or only partly - in a crystalline arrangement can be studied at high resolution. This will be achieved by developing certain image processing algorithms and tools to apply iterative refine-ment procedures to 3D structure reconstructions, and by adopting some of the recent algorithmic develop-ments in the single particle field such as gold-standard FSC for reliable resolution estimation or drift-correction for dose-fractionation into the 2dx electron crystallography software suite. Finally, we will imple-ment an interface of the 2dx package to leading single particle software suites such as RELION and/or FREALIGN, and adapt the latter to the specific geometries and settings of cryo-EM data of membrane-embedded membrane proteins. For development purposes, a large high-resolution dataset of images of a potassium channel membrane protein system is available. These images are recorded of small 2D crystals that diffract only to about 12 Å resolution. Preliminary application of novel algorithms has already shown significant resolution improve-ments, so that the helical pitch of alpha helices and some side-chain densities are recognizable in the 3D reconstruction. This software will continue being available open-source under the GPL at 2dx.org, and we will organize regu-lar workshops for knowledge transfer and planning of further development targets.
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