Project

Back to overview

Electron Microscopy of Membrane Proteins

English title Electron Microscopy of Membrane Proteins
Applicant Stahlberg Henning
Number 146929
Funding scheme Project funding (Div. I-III)
Research institution C-CINA Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Biophysics
Start/End 01.04.2013 - 31.03.2016
Approved amount 605'644.00
Show all

Keywords (5)

potassium channel; 2D crystals; membrane protein structure; electron crystallography; electron tomography

Lay Summary (German)

Lead
Membran Proteine gehören zu den wichtigsten molekularen Maschinen, welche wir in unseren menschlichen Körpern haben. Ihr korrektes Funktionieren ist eine Voraussetzung für gesunde menschliche Zellen. Dafür ist die Kenntniss der atomaren Struktur der Membran Proteine essentiell, so wie sie in der Regel durch Röntenstrukturuntersuchungen bestimmt werden kann.
Lay summary

In diesem Projekt werden wir die 3D Struktur von ausgewählten Membran Proteinen untersuchen, während die Proteine in biologischen Lipid Membranen eingebaut sind. Interessant dabei wird sein, dass wir die Struktur der Proteine in Membranen bestimmen wollen, welche lebensnahe Bedingungen erleben, indem auf beiden Seiten der Membranen unterschiedliche flüssige Lösungen vorherschen. Um dies zu erreichen, werden wir die Proteine in die Membranen von Lipid Vesikeln einbauen, was kleine Membran-Bläschen sind. Diese Vesikel werden im inneren und im äusseren unterschiedliche Lösungen haben, so wie dies in Membranen in natürlichen Zellen auch der Fall ist. Die Membran Proteine, welche wir untersuchen werden, sind Kaliumkanäle, Natrium-Zitrat Symporter, sowie ein Komplex aus einem bakteriellen Protein mit einem Virus Dock-Protein. 


Direct link to Lay Summary Last update: 05.05.2013

Lay Summary (English)

Lead
Membrane proteins in our human body are among the most important molecular machines for human health. A detailed mechanistic understanding of their function requires not only a high-resolution structure as usually obtained by X-ray diffraction methods, but also information about the structure of these membrane proteins under life-like conditions.
Lay summary

Here, we will study the 3D structure of membrane proteins, while they are embedded in biological lipidic membranes. Importantly, we will seek to study the structure of these proteins under life-like environmental conditions, by placing the membrane proteins into the membrane of lipid vesicles (tiny bags) that have different buffer solutions on the inside than on the outside. This should allow studying the reaction of the membrane proteins to gradients in the buffer solutions on both sides of the membranes.  Systems to study are potassium channels, sodium-citrate symporters, and a complex from a bacterial protein with a virus docking protein. 


Direct link to Lay Summary Last update: 05.05.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins.
Abeyrathne Priyanka D, Chami Mohamed, Stahlberg Henning (2016), Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins., in Biochimie, 128-129, 154-62.
Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP.
Lauer Matthias E, Graff-Meyer Alexandra, Rufer Arne C, Maugeais Cyrille, von der Mark Elisabeth, Matile Hugues, D'Arcy Brigitte, Magg Christine, Ringler Philippe, Müller Shirley A, Scherer Sebastian, Dernick Gregor, Thoma Ralf, Hennig Michael, Niesor Eric J, Stahlberg Henning (2016), Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP., in Journal of structural biology, 194(2), 191-8.
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, 30111-3.
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.
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.
Structure of the type VI secretion system contractile sheath.
Kudryashev Mikhail, Wang Ray Yu-Ruei, Brackmann Maximilian, Scherer Sebastian, Maier Timm, Baker David, DiMaio Frank, Stahlberg Henning, Egelman Edward H, Basler Marek (2015), Structure of the type VI secretion system contractile sheath., in Cell, 160(5), 952-62.
Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation.
Kudryashev Mikhail, Diepold Andreas, Amstutz Marlise, Armitage Judith P, Stahlberg Henning, Cornelis Guy R (2015), Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation., in Molecular microbiology, 95(5), 875-84.
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.
Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports.
Kowal Justyna Ł, Kowal Julia K, Wu Dalin, Stahlberg Henning, Palivan Cornelia G, Meier Wolfgang P (2014), Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports., in Biomaterials, 35(26), 7286-94.
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.
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.
In situ structural analysis of the Yersinia enterocolitica injectisome.
Kudryashev Mikhail, Stenta Marco, Schmelz Stefan, Amstutz Marlise, Wiesand Ulrich, Castaño-Díez Daniel, Degiacomi Matteo T, Münnich Stefan, Bleck Christopher Ke, Kowal Julia, Diepold Andreas, Heinz Dirk W, Dal Peraro Matteo, Cornelis Guy R, Stahlberg Henning (2013), In situ structural analysis of the Yersinia enterocolitica injectisome., in eLife, 2, 00792-00792.
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.

Collaboration

Group / person Country
Types of collaboration
Cecile Breyton, IBS, Grenoble France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
Gunnar Schröder, FZ Jülich Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Crina Nimigean, Cornell University, NY 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
Workshop on Sub-volume averaging with Dynamo 25.08.2015 Basel, Switzerland
Workshop on Sub-volume averaging with Dynamo 18.08.2014 Basel, Switzerland
GRC on 3DEM (Chair) 22.06.2014 Girona, Spain, Spain
Workshop on Sub-volume Averaging with Dynamo 19.08.2013 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)
127545 Electron Microscopy of Ion Channels 01.02.2010 Project funding (Div. I-III)
164074 Cryo-Electron Microscopy in the ZMB of the University of Basel 01.06.2016 R'EQUIP

Abstract

Membrane proteins are central to health and disease. A detailed understanding of the function of membrane proteins requires access to high-resolution structural data and mechanistic insight into conformational changes occurring when the proteins are active. We here propose to employ the recently established cryo-electron microscopy imaging capabilities in C-CINA, the Center for Cellular Imaging and NanoAnalytics, and our newly developed image processing software, to study the dynamic structure of selected membrane protein systems in liposomes, a close-to-native environment: Vesicles containing excellently ordered two-dimensional (2D) crystalline membrane protein arrays will be vitrified in aqueous buffer solutions, whereby different buffer solutions will be established on the inside and outside of the liposomes. The resulting buffer gradient will impose an electric potential, pH and/or ligand concentration gradient across the membranes, which will be experienced by the reconstituted proteins. Further, 2D crystals grown with a 180-degree screw-axis symmetry will place adjacent membrane proteins in alternating orientations in the membranes, allowing us to determine the 3D structures of oppositely regulated states in one experiment.We will employ a combination of electron crystallography (using our 2dx software), electron tomography (using our Dynamo software), and single particle processing of 2D crystals (using our new module in the 2dx software), to determine the 3D structure at 7Å resolution or better of selected systems. We already have excellently ordered 2D crystals of the bacterial iron-ferrichrome transporter FhuA decorated with the bacteriophage T5 tip protein pb5, of the cAMP-modulated potassium channel MloK1 from M. loti, and of the sodium/citrate symporter CitS from K. pneumonia, and we will grow 2D crystals of the voltage gated potassium channel KvAP from A. pernix. The exceptional quality and contrast of the FhuA-pb5 crystals will yield a high-resolution structure and allow our new methodology to be finely tuned. Analysis of the CitS crystals will reveal the active site and document a new, unknown fold of an important class of membrane proteins. We will expose MloK1 crystals to varying cAMP concentrations on both sides of the lipid membrane, and KvAP crystals to a Nernst electrical potential difference to trigger voltage gating, and document the conformational changes. The produced data will complement existing high-resolution X-ray structural studies with information about the dynamic functioning of these classes of membrane proteins. Importantly, this work will, for the first time, allow the direct structural observation of the voltage gating mechanism of a voltage-gated potassium channel.
-