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Software for Electron Microscopy of Membrane Proteins

Applicant Stahlberg Henning
Number 144427
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 01.10.2012 - 31.03.2016
Approved amount 445'981.00
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All Disciplines (2)

Discipline
Other disciplines of Physics
Biophysics

Keywords (6)

membrane protein; 2D crystal; image processing; electron crystallography; maximum likelihood; single particle

Lay Summary (English)

Lead
Membrane proteins are central to health and disease, and represent the major focus of intensive research efforts. The structure of two-dimensionally crystallized membrane proteins can be determined by electron microscopy and image processing. We develop a software package for this task, called 2dx, which we extend to improve resolution and speed of the structure determination.
Lay summary

Background:

The structure determination of human membrane proteins from electron microscopy images usually requires the proteins to be available in well-ordered two-dimensional crystals (side-by-side densely packed in a membrane). These crystals are imaged in the electron microscope from different viewing angles, and the 3D structure of the proteins is reconstructed through a combination of images from different viewing angles.

So far, this required images of perfectly ordered 2D crystals that were prepared on extremely flat supports.

Aims:

Here we will extend the 2dx software package by implementing a single particle-based approach, where we treat the images as collection of particles, instead of crystals. This allows to refine the individual location and orientation of each membrane protein particle, thereby giving access to a higher resolution of the final reconstruction.

Significance:

The software will be available as open-source software in our 2dx package, which now enjoys over 600 external users from life sciences and materials sciences electron microscopy fields (http://2dx.org). As such, the algorithms will immediately be available in a user-friendly and heavily used environment to other research labs. 


Direct link to Lay Summary Last update: 07.11.2012

Responsible applicant and co-applicants

Employees

Publications

Publication
Dynamo Catalogue: Geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms.
Castaño-Díez Daniel, Kudryashev Mikhail, Stahlberg Henning (2016), Dynamo Catalogue: Geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms., in Journal of structural biology, 30111.
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death.
Sborgi Lorenzo, Rühl Sebastian, Mulvihill Estefania, Pipercevic Joka, Heilig Rosalie, Stahlberg Henning, Farady Christopher J, Müller Daniel J, Broz Petr, Hiller Sebastian (2016), GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death., in The EMBO journal, 35(16), 1766-78.
Structure of the T4 baseplate and its function in triggering sheath contraction.
Taylor Nicholas M I, Prokhorov Nikolai S, Guerrero-Ferreira Ricardo C, Shneider Mikhail M, Browning Christopher, Goldie Kenneth N, Stahlberg Henning, Leiman Petr G (2016), Structure of the T4 baseplate and its function in triggering sheath contraction., in Nature, 533(7603), 346-52.
The lipidome associated with the γ-secretase complex is required for its integrity and activity.
Ayciriex Sophie, Gerber Hermeto, Osuna Guillermo M Garcia, Chami Mohamed, Stahlberg Henning, Shevchenko Andrej, Fraering Patrick C (2016), The lipidome associated with the γ-secretase complex is required for its integrity and activity., in The Biochemical journal, 473(3), 321-34.
The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles.
Kudryashev Mikhail, Castaño-Díez Daniel, Deluz Cédric, Hassaine Gherici, Grasso Luigino, Graf-Meyer Alexandra, Vogel Horst, Stahlberg Henning (2016), The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles., in Structure (London, England : 1993), 24(1), 165-70.
3D reconstruction of two-dimensional crystals.
Stahlberg Henning, Biyani Nikhil, Engel Andreas (2015), 3D reconstruction of two-dimensional crystals., in Archives of biochemistry and biophysics, 581, 68-77.
CTF Challenge: Result summary.
Marabini Roberto, Carragher Bridget, Chen Shaoxia, Chen James, Cheng Anchi, Downing Kenneth H, Frank Joachim, Grassucci Robert A, Bernard Heymann J, Jiang Wen, Jonic Slavica, Liao Hstau Y, Ludtke Steven J, Patwari Shail, Piotrowski Angela L, Quintana Adrian, Sorzano Carlos O S, Stahlberg Henning, Vargas Javier, Voss Neil R, Chiu Wah, Carazo Jose M (2015), CTF Challenge: Result summary., in Journal of structural biology, 190(3), 348-59.
2dx_automator: implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline.
Scherer Sebastian, Kowal Julia, Chami Mohamed, Dandey Venkata, Arheit Marcel, Ringler Philippe, Stahlberg Henning (2014), 2dx_automator: implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline., in Journal of structural biology, 186(2), 302-7.
A KcsA/MloK1 chimeric ion channel has lipid-dependent ligand-binding energetics.
McCoy Jason G, Rusinova Radda, Kim Dorothy M, Kowal Julia, Banerjee Sourabh, Jaramillo Cartagena Alexis, Thompson Ameer N, Kolmakova-Partensky Ludmila, Stahlberg Henning, Andersen Olaf S, Nimigean Crina M (2014), A KcsA/MloK1 chimeric ion channel has lipid-dependent ligand-binding energetics., in The Journal of biological chemistry, 289(14), 9535-46.
Cryo-electron microscopy of membrane proteins.
Goldie Kenneth N, Abeyrathne Priyanka, Kebbel Fabian, Chami Mohamed, Ringler Philippe, Stahlberg Henning (2014), Cryo-electron microscopy of membrane proteins., in Methods in molecular biology (Clifton, N.J.), 1117, 325-41.
Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1.
Kowal Julia, Chami Mohamed, Baumgartner Paul, Arheit Marcel, Chiu Po-Lin, Rangl Martina, Scheuring Simon, Schröder Gunnar F, Nimigean Crina M, Stahlberg Henning (2014), Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1., in Nature communications, 5, 3106-3106.
Single particle 3D reconstruction for 2D crystal images of membrane proteins.
Scherer Sebastian, Arheit Marcel, Kowal Julia, Zeng Xiangyan, Stahlberg Henning (2014), Single particle 3D reconstruction for 2D crystal images of membrane proteins., in Journal of structural biology, 185(3), 267-77.
The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography.
Kudryashev Misha, Aktoudianaki Aikaterini, Dedoglou Dimitrios, Stahlberg Henning, Tsiotis Georgios (2014), The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography., in Biochimica et biophysica acta, 1837(10), 1635-42.
X-ray structure of the mouse serotonin 5-HT3 receptor.
Hassaine Ghérici, Deluz Cédric, Grasso Luigino, Wyss Romain, Tol Menno B, Hovius Ruud, Graff Alexandra, Stahlberg Henning, Tomizaki Takashi, Desmyter Aline, Moreau Christophe, Li Xiao-Dan, Poitevin Frédéric, Vogel Horst, Nury Hugues (2014), X-ray structure of the mouse serotonin 5-HT3 receptor., in Nature, 512(7514), 276-81.
Automation of image processing in electron crystallography.
Arheit Marcel, Castaño-Díez Daniel, Thierry Raphaël, Gipson Bryant R, Zeng Xiangyan, Stahlberg Henning (2013), Automation of image processing in electron crystallography., in Methods in molecular biology (Clifton, N.J.), 955, 313-30.
Image processing of 2D crystal images.
Arheit Marcel, Castaño-Díez Daniel, Thierry Raphaël, Gipson Bryant R, Zeng Xiangyan, Stahlberg Henning (2013), Image processing of 2D crystal images., in Methods in molecular biology (Clifton, N.J.), 955, 171-94.
Merging of image data in electron crystallography.
Arheit Marcel, Castaño-Diéz Daniel, Thierry Raphaël, Abeyrathne Priyanka, Gipson Bryant R, Stahlberg Henning (2013), Merging of image data in electron crystallography., in Methods in molecular biology (Clifton, N.J.), 955, 195-209.
Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism.
Degiacomi Matteo T, Iacovache Ioan, Pernot Lucile, Chami Mohamed, Kudryashev Misha, Stahlberg Henning, van der Goot F Gisou, Dal Peraro Matteo (2013), Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism., in Nature chemical biology, 9(10), 623-9.
Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography.
Kebbel Fabian, Kurz Mareike, Arheit Marcel, Grütter Markus G, Stahlberg Henning (2013), Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography., in Structure (London, England : 1993), 21(7), 1243-50.
Structure of the dodecameric Yersinia enterocolitica secretin YscC and its trypsin-resistant core.
Kowal Julia, Chami Mohamed, Ringler Philippe, Müller Shirley A, Kudryashev Mikhail, Castaño-Díez Daniel, Amstutz Marlise, Cornelis Guy R, Stahlberg Henning, Engel Andreas (2013), Structure of the dodecameric Yersinia enterocolitica secretin YscC and its trypsin-resistant core., in Structure (London, England : 1993), 21(12), 2152-61.
Thermal unfolding of a mammalian pentameric ligand-gated ion channel proceeds at consecutive, distinct steps.
Tol Menno B, Deluz Cédric, Hassaine Gherici, Graff Alexandra, Stahlberg Henning, Vogel Horst (2013), Thermal unfolding of a mammalian pentameric ligand-gated ion channel proceeds at consecutive, distinct steps., in The Journal of biological chemistry, 288(8), 5756-69.
Assessing the benefits of focal pair cryo-electron tomography.
Kudryashev Mikhail, Stahlberg Henning, Castaño-Díez Daniel (2012), Assessing the benefits of focal pair cryo-electron tomography., in Journal of structural biology, 178(2), 88-97.
Characterization of the motion of membrane proteins using high-speed atomic force microscopy.
Casuso Ignacio, Khao Jonathan, Chami Mohamed, Paul-Gilloteaux Perrine, Husain Mohamed, Duneau Jean-Pierre, Stahlberg Henning, Sturgis James N, Scheuring Simon (2012), Characterization of the motion of membrane proteins using high-speed atomic force microscopy., in Nature nanotechnology, 7(8), 525-9.
Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments.
Castaño-Díez Daniel, Kudryashev Mikhail, Arheit Marcel, Stahlberg Henning (2012), Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments., in Journal of structural biology, 178(2), 139-51.
Limiting factors in single particle cryo electron tomography.
Kudryashev Mikhail, Castaño-Díez Daniel, Stahlberg Henning (2012), Limiting factors in single particle cryo electron tomography., in Computational and structural biotechnology journal, 1, 201207002-201207002.
Projection structure of the secondary citrate/sodium symporter CitS at 6 Å resolution by electron crystallography.
Kebbel Fabian, Kurz Mareike, Grütter Markus G, Stahlberg Henning (2012), Projection structure of the secondary citrate/sodium symporter CitS at 6 Å resolution by electron crystallography., in Journal of molecular biology, 418(1-2), 117-26.

Collaboration

Group / person Country
Types of collaboration
Prof. Xiangyan Zeng, FVS University United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Crina Nimigean, Cornell Univ., NY, USA United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Scientific events



Self-organised

Title Date Place
EMBO Workshop on Cryo-EM (Co-Organizer) 28.08.2016 EMBL, Heidelberg, Germany, Germany
Workshop on Image Processing with 2dx (Organizer) 22.08.2016 University of Basel, Switzerland
Cryo-EM 3D Image Analysis Symposium 2016 (Co-Organizer) 30.03.2016 Tahoe, CA, USA, United States of America
EMBO Practical Course on 3DEM (Co-Organizer) 31.08.2014 EMBL, Heidelberg, Germany
GRC on 3DEM (Chair) 22.06.2014 Girona, Spain, Spain
GRC on 3DEM (Vice-Chair) 23.06.2013 New London, NH, USA, United States of America

Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) 2dx Website Website International 2012

Associated projects

Number Title Start Funding scheme
166164 High-Resolution Membrane Protein Structures by Cryo-EM 01.04.2016 Project funding (Div. I-III)
146929 Electron Microscopy of Membrane Proteins 01.04.2013 Project funding (Div. I-III)
126490 Software for Electron Crystallography 01.10.2009 Project funding (Div. I-III)

Abstract

Membrane proteins are central to health and disease. Electron crystallography can deter-mine the high-resolution structure of membrane proteins from membrane-reconstituted and two-dimensionally crystallized protein. The Transmission Electron Microscope (TEM) is used to study membrane proteins in the lipid membrane environment, which is a powerful tool to study the structure and dynamic of membrane proteins, and of membrane protein complexes that still resist high-resolution 3D crystallization or large-quantity expression efforts.Two-dimensional membrane crystals are imaged by cryo-electron microscopy (cryo-EM). The high-resolution 3D membrane protein structure can then be reconstructed by computer im-age processing. Until recently, the only available software for that task was the MRC software (Crowther et al., 1996). Starting in 2003, my group has created a software system, called 2dx, which embeds the MRC software into a user-friendly graphical user interface, and in addition offers a multitude of additional functions that enable full automation of the processing task, user guidance, interactive documentation and help, and allows automated iterative refinement of the processing results. 2dx received good acceptance in and excellent feedback from the electron crystallography community, and we now count more than 240 external users (as of 1/15/2009).We here propose to extend the 2dx software by new algorithms, to increase resolution and speed of the membrane protein structure determination by electron crystallography, and to en-able high-resolution structure determination also from badly ordered 2D crystals. We will imple-ment a single-particle approach for 3D reconstructions from tilted 2D crystal images, and extend this by a maximum likelihood algorithm. We will develop a hybrid input output algorithm imple-mentation to improve the Z resolution of electron crystallography, and to reduce the need to record images of highly tilted 2D crystals. We will also modernize 2dx, by enabling cluster CPU and cluster GPU computing, and interface it with other systems like Eman2 and IPLT.
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