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Grünewald Group

Structural Cell Biology of Viruses

Prof. Dr. Kay Grünewald

Group Leader, Facility Director (ALFM and Cryo-EM)

+49 40 8998 87700
kay.gruenewald@cssb-hamburg.de

Home Institute

Heinrich Pette Institute, Leibniz Institute for Experimental Virology
www.hpi-hamburg.de

Universität Hamburg
www.uni-hamburg.de

We have pioneered the application of cryoET to isolated pleomorphic viruses revealing their three-dimensional supramolecular organization. Examples are virus particle structures for Herpes simplex virus, HIV-1 and Bunyaviruses. We next concentrated on the cell biology of virus infection. Understanding the entirety of a virus’ ‘life cycle’ requires an understanding of its transient structures at the molecular level.

Research Projects

Previous and Current Research

Cells constitute the smallest autonomous units of life. Supramolecular complexes carry out essentially all functions and processes and form the cells structural elements. The tightly regulated structural and functional organization of a cell at this level is currently only rudimentary understood. A comprehensive analysis of this organization and its dynamic changes requires tools that allow for studying these complexes in their native environment. We apply electron cryo tomography (cryoET) in combination with other techniques to approach selected aspects of this highly ordered network analysing protein complexes in situ. Sample preparation by fast vitrification, i.e. embedding in amorphous ice, ensures excellent preservation of structure down to the atomic level.

We have pioneered the application of cryoET to isolated pleomorphic viruses revealing their three-dimensional supramolecular organization. Examples are virus particle structures for Herpes simplex virus, HIV-1 and Bunyaviruses. We next concentrated on the cell biology of virus infection. Understanding the entirety of a virus’ ‘life cycle’ requires an understanding of its transient structures at the molecular level. The aim is a comprehensive picture of the functional interaction between viral protein complexes and cellular structures in the course of the infection. Viruses also serve as dedicated tools to mine the molecular detail of cellular tomograms. Being able to enter cells via physiological pathways and being recognizable among the multitude of other structural features inside the host's cytoplasm, viruses allow for following dynamic cellular processes.

Electron cryo microscopy provides an excellent platform for interfacing with other approaches, like biochemical and X-ray crystallographic studies, and the integration of those inputs with native sub-cellular structural information. Driven by our biological questions we are involved in various efforts of methods development including the combination of cellular and molecular cryoET with ‘single particle’ approaches, advanced fluorescence (cryo) microscopy and soft X-ray cryo microscopy/tomography in a correlative fashion, but also in integrating better with protein-protein-interaction data (e.g. from mass spectrometry) and molecular dynamics simulations.

Research Team

24

Group Leader

Prof. Dr. Kay Grünewald
Phone:+49 40 8998 87700

E-Mail

Project Management

Dr. Frederike Ahr
Phone:+49 40 8998 87703

E-Mail

Senior Scientist

Dr. Lindsay Baker, Oxford

Subgroup Leader

Dr. Jens B. Bosse

Staff Scientist Cryo EM

Cornelia Cazey
Phone:+49 40 8998 87706

E-Mail

Postdoctoral Scientist

Dr. Josie Fereira

E-Mail

PhD Student

Felix Johann Flomm
Phone:+49 40 8998 87708

E-Mail

Postdoctoral Scientist

Dr. Linda Franken
Phone:+49 40 8998 87709

E-Mail

Senior Scientist

Dr. Christoph Hagen, Deputy
Phone:+49 40 8998 87702

E-Mail

Team Assistant

Débora Lage
Phone:+49 40 8998 87701

E-Mail

Postdoctoral Scientist

Dr. Emily Machala

PhD Student

Søren Pfitzner
Phone:+49 40 8998 87709

E-Mail

PhD Student

Vojtech Prazak
Phone:+49 40 8998 87709

E-Mail

Postdoctoral Scientist

Dr. Emmanuelle Quemin
Phone:+49 40 8998 87708

E-Mail

HPI Tech. Platform

Dr. Rudolph Reimer, Co-Head

PhD Student

Rene Rosch
Phone:+49 40 8998 87708

E-Mail

PhD Student

Karin Ruban
Phone:+49 40 8998 87711

E-Mail

Lab manager

Julia Sandberg

Technical Assistant

Carola Schneider, HPI

Core Facility Head

Dr. Carolin Seuring
Phone:+49 40 8998 87707

E-Mail

Postdoctoral Scientist

Dr. Timothy Soh
Phone:+49 40 8998 87711

E-Mail

PhD Student

Sigurdur Thorkelsson
Phone:+49 40 8998 87711

E-Mail

Senior Scientist

Dr. Daven Vasishtan

Postdoctoral Scientist

Dr. Benjamin Vollmer
Phone:+49 40 8998 87709

E-Mail

Publications

2019

Hernández Durán A, Greco TM, Vollmer B, Cristea IM, Grünewald K, Topf M (2019) Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships.PLoS Biol.17(6):e3000316.

Moser F, Pražák V, Mordhorst V, Andrade DM, Baker LA, Hagen C, Grünewald K, Kaufmann R (2019) Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy.  Proc Natl Acad Sci USA. 116(11):4804-4809.

2018

Chorev DS, Baker LA, Wu D, Beilsten-Edmands V, Rouse SL, Zeev-Ben-Mordehai T, Jiko C, Samsudin F, Gerle C, Khalid S, Stewart AG, Matthews SJ, Grünewald K, Robinson CV (2018) Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry.Science. 362(6416):829-834.

Kaufmann R, Grünewald K (2018) Super-resolution CLEM: from room temperature to cryo-imaging, in ‘The 2018 correlative microscopy techniques roadmap’ J. Physics D: Appl. Physics 51(443001): 9-10. https://doi.org/10.1088/1361-6463/aad055

Grünewald K, Krey T (2018) Virus structure and expression - Editorial Overview. Curr. Opin. Virol. 31: iii-v. doi: 10.1016/j.coviro.2018.09.006.

Baker LA, Sinnige T, Schellenberger P, de Keyzer J, Siebert CA, Driessen AJM, Baldus M, Grünewald K (2018) Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments. Structure 26(1): 161-170.e3. https://doi.org/10.1016/j.str.2017.11.011.

2017

Baker LA, Grange M, Grünewald K (2017) Electron cryo-tomography captures macromolecular complexes in native environments. Curr Opin Struct Biol. 46: 149-156. https://doi.org/10.1016/j.sbi.2017.08.005.

Patwardhan A, Brandt R, Butcher SJ, Collinson L, Gault D, Grünewald K, Hecksel C, Huiskonen JT, Iudin A, Jones ML, Korir PK, Koster AJ, Lagerstedt I, Lawson CL, Mastronarde D, McCormick M, Parkinson H, Rosenthal PB, Saalfeld S, Saibil HR, Sarntivijai S, Solanes Valero I, Subramaniam S, Swedlow JR, Tudose I, Winn M, Kleywegt GJ (2017) Building bridges between cellular and molecular structural biology. Elife 6. pii: e25835

Clare DK, Siebert CA, Hecksel C, Hagen C, Mordhorst V, Grange M, Ashton AW, Walsh MA, Grünewald K, Saibil HR, Stuart DI, Zhang P (2017) Electron Bio-Imaging Centre (eBIC): the UK national research facility for biological electron microscopy. Acta Cryst. D73, 488–495.

Montespan C, Marvin SA, Austin S, Burrage AM, Roger B, Rayne F, Faure M, Campell EM, Schneider C, Reimer R, Grünewald K, Wiethoff CM, Wodrich H (2017) Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid. PLoS Pathog. 13(2):e1006217.

2016

Stoeber M, Schellenberger P, Siebert CA, Leyrat C, Helenius A, Grünewald K (2016) Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs. PNAS 113(50): E8069-E8078.

Grange M, Vasishtan D, Grünewald K (2016) Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes. J. Struct. Biol. S1047-8477(16) 30139-3 50

Riedel C, Vasishtan D, Siebert CA, Whittle C, Lehmann MJ, Mothes W, Grünewald K (2016) Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell. J. Struct. Biol. S1047-8477(16)30123-X.

Ashford P, Hernandez A, Greco TM, Buch A, Sodeik B, Cristea IM, Grünewald K, Shepherd A, Topf M (2016) HVint: A strategy for identifying novel protein-protein interactions in herpes simplex virus type 1. Mol Cell Proteomics. 15(9):2939-53.

Wolff G, Hagen C, Grünewald K, Kaufmann R (2016) Towards correlative super-resolution fluorescence and electron cryo-microscopy. Biol. Cell. 2016 Sep;108(9):245-58.

Zeev-Ben-Mordehai T, Vasishtan D, Hernández Durán A, Vollmer B, White P, Prasad Pandurangan Siebert, CA Topf M, Grünewald K (2016) Two distinct trimeric conformations of natively membrane-anchored full-length Herpes simplex virus 1 glycoprotein B. PNAS 113(15): 4176-4181.

2015

Zeev-Ben-Mordehai T, Weberruß M, Lorenz M, Cheleski J, Hellberg T, Whittle C, El Omari K, Vasishtan D, Dent KC, Harlos K, Franzke K, Hagen C, Klupp BG, Antonin W, Mettenleiter TC, Grünewald K (2015) Crystal structure of the herpesvirus nuclear egress complex provides insights into inner nuclear membrane remodeling. Cell Reports 13: 2645–2652.

Hagen C, Dent KC, Zeev-Ben-Mordehai T, Grange M, Bosse JB, Whittle C, Klupp BG, Siebert CA, Vasishtan D, Bäuerlein FJB, Cheleski J, Werner S, Guttmann P, Rehbein S, Henzler K, Demmerle J, Adler B, Koszinowski U, Schermelleh L, Schneider G, Enquist LW, Plitzko JP, Mettenleiter TC, Grünewald K (2015) Structural Basis of Vesicle Formation at the Inner Nuclear Membrane. Cell 163: 1692–1701.

Sali A, Berman HM, Schwede T, Trewhella J, Kleywegt G, Burley SK, Markley J, Nakamura H, Adams P, Bonvin AM, Chiu W, Peraro MD, Di Maio F, Ferrin TE, Grünewald K, Gutmanas A, Henderson R, Hummer G, Iwasaki K, Johnson G, Lawson CL, Meiler J, Marti-Renom MA, Montelione GT, Nilges M, Nussinov R, Patwardhan A, Rappsilber J, Read RJ, Saibil H, Schröder GF, Schwieters CD, Seidel CA, Svergun D, Topf M, Ulrich EL, Velankar S, Westbrook JD (2015) Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop. Structure 23: 1156-1167.

Johnson E, Seiradake E, Jones EY, Davis I, Grünewald K, Kaufmann R (2015) Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Scientific Reports 5: 9583.

Martinez R, Schellenberger P, Vasishtan D, Aknin C, Austin S, Dacheux D, Rayne F, Siebert A, Ruzsics Z, Grünewald K, Wodrich H (2015) The amphipatic helix of adenovirus capsid protein VI contributes to penton release and post entry sorting. J. Virol. 89: 2121-35

Saibil HR, Grünewald K, Stuart DI (2015) A national facility for biological cryo-electron microscopy. Acta Crystal. D 71: 127-135. 51

2014

Zeev-Ben-Mordehai T, Vasishtan D, Siebert A, Whittle C, Grünewald K (2014) Extracellular vesicles: a platform for the structure determination of membrane proteins by cryoEM. Structure 22:1687-1692.

Anderson F., Savulescu A., Rudolph K., Schipke J., Cohen I., Ibiricu I., Rotem A., Grünewald K., Sodeik B., Harel A. 2014. Targeting of viral capsids to nuclear pores in a cell-free reconstitution system. Traffic 15: 1266- 1281.

Patwardhan A, Ashton A, Brandt R, Butcher S, Carzaniga R, Chiu W, Collinson L, Doux P, Duke E, Ellisman M, Franken E, Grünewald K, Heriche J-K, Koster A, Kühlbrandt W, Lagerstedt I, Larabell C, Lawson CL, Saibil HR, Sanz-García E, Subramaniam S, Verkade P, Swedlow JR, Kleywegt GJ (2014) A 3D cellular context for the macromolecular world. Nat. Struct. Mol. Biol. 21(10): 841-845.

Dent KC, Hagen C, Grünewald K (2014) Critical step-by-step approaches toward correlative fluorescence/soft X-ray cryo-microscopy of adherent mammalian cells. Meth. Cell Biol. 24: 179-216.

Hagen C, Werner S, Carregal-Romero S, Malhas AN, Klupp BG, Guttmann P, Rehbein S, Henzler K, Mettenleiter TC, Vaux DJ, Parak WJ, Schneider G, Grünewald K (2014) Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryomicroscopy/ tomography of nucleoplasmic reticulum and apoptosis in mammalian cells. Ultramicroscopy 146: 46-54.

Kaufmann R, Schellenberger P, Seiradake E, Dobbie IM, Jones EY, Davis I, Hagen C, Grünewald K (2014) Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions. Nano Letters 4(7): 4171-4175.

Zeev-Ben-Mordehai T, Vasishtan D, Siebert A, Grünewald K (2014) The full-length cell-cell fusogen EFF-1 is monomeric and upright to the membrane. Nature Comm. 5: 3912.

Kaufmann R., Hagen C., Grünewald K. 2014. Fluorescence cryomicroscopy: current challenges and prospects. Curr. Opin. Chem. Biol. 20: 86–91.

Stauffer S, Rahman S, de Marco A, Carlson LA, Glass B, Oberwinkler H, Herold N, Briggs J, Müller B, Grünewald K, Kräusslich HG (2014) The NC domain of Gag is dispensable for actin incorporation into HIV-1 and for association of viral budding sites with cortical F-actin. J. Virol. 88(14): 7893-7903.

Koster AJ, Grünewald K (2014) Editorial on Correlative Microscopy Special Issue. Ultramicroscopy 143: 1-2.

Schellenberger P, Kaufmann R, Siebert CA, Hagen C, Wodrich H, Grünewald K (2014) High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers. Ultramicroscopy 143: 41-45.52

Zeev-Ben-Mordehai T, Hagen C, Grünewald K (2014) A cool hybrid approach to the herpesvirus ‘life’ cycle Curr. Opin. Virol. 5: 42–49.

2013

Maurer UE, Zeev-Ben-Mordehai T, Pandurangan AP, Cairns TM, Hannah BP, Whitbeck JC, Eisenberg RJ, Cohen GH, Topf M, Huiskonen JT, Grünewald K (2013) The structure of herpesvirus fusion glycoprotein B-bilayer complex reveals the protein-membrane and lateral protein-protein interaction. Structure 21: 1396-1405.

Ibiricu I, Maurer UE, Grünewald K (2013) Characterization of herpes simplex virus type 1 L-particle assembly and egress in hippocampal neurons by electron cryotomography. Cell. Microbiol. 15(2): 285-291.

2012

Hagen C, Guttmann P, Klupp B, Werner S, Rehbein S, Mettenleiter TC, Schneider G, Grünewald K (2012) Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J. Struct. Biol. 177: 193-201.

2011

Ibiricu I, Huiskonen JT, Döhner K, Bradke F, Sodeik B, Grünewald K (2011) Cryo Electron Tomography of Herpes Simplex Virus during Axonal Transport and Secondary Envelopment in Primary Neurons. PLoS Pathog. 7: e1002406.

Karotki L, Huiskonen JT, Stefan CJ, Ziółkowska NE, Roth R, Surma MA, Krogan NJ, Emr SD, Heuser J, Grünewald K, Walther TC (2011) Eisosome Proteins Assemble into a Membrane Scaffold. Journal of Cell Biology 195: 889–902.

Avinoam O, Fridman K, Valansi C, Abutbul I, Zeev-Ben-Mordehai T, Maurer UE, Sapir A, Danino D, Grünewald K, White JM, Podbilewicz B (2011) Conserved eukaryotic fusogens can fuse viral envelopes to cells. Science 332: 589-592.

2010

Carlson LA, de Marco A, Oberwinkler H, Habermann A, Briggs JA, Kräusslich HG, Grünewald K (2010) Cryo electron tomography of native HIV-1 budding sites. PLoS Pathog. 6: e1001173.

Brandt F, Carlson LA, Hartl FU, Baumeister W, Grünewald K (2010) The three-dimensional organization of polyribosomes in intact human cells. Mol. Cell 39: 560-569.

Huiskonen JT, Hepojoki J, Laurinmäki P, Vaheri A, Lankinen H, Butcher SJ, Grünewald K (2010) Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses. J. Virol. 84: 4889-4897.

Scrivano L, Esterlechner J, Mühlbach H, Ettischer N, Hagen C, Grünewald K, Mohr CA, Ruzsics Z, Koszinowski U, Adler B (2010) The m74 gene product of murine cytomegalovirus (MCMV) is a functional homolog of human CMV gO and determines the entry pathway of MCMV. J. Virol. 84: 4469-4480.53

Grünewald K (2010) Viral fusion: how Flu induces dimples on liposomes. EMBO J. 29: 1165-1166.

2009

Grünewald K (2009) Adding a spatial dimension to the proteome. Nature Methods 6: 798-800.

Kopp SJ, Banisadr G, Glajch K, Maurer UE, Grünewald K, Miller RJ, Osten P Spear, PG (2009) Infection of neurons and encephalitis after intracranial inoculation of herpes simplex virus requires the entry receptor nectin-1. PNAS 106: 17916-17920.

Huiskonen JT, Overby AK, Weber F, Grünewald K (2009) Electron Cryo- Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for GN-GC Glycoprotein Heterodimers. J. Virol. 83: 3762-3769.

2008

Carlson LA, Briggs JA, Glass B, Riches JD, Simon MN, Johnson MC, Müller B, Grünewald K, Kräusslich HG (2008) Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis. Cell Host Microbe 4: 592-599.

Maurer UE, Sodeik B, Grünewald K (2008) Intermediates of membrane fusion in herpesvirus entry captured by cryo electron tomography. PNAS 105(30): 10559-10564.

Hagen C, Grünewald K (2008) Microcarriers for high-pressure freezing and cryosectioning of adherent cells. J. Microsc. 30(2): 288-296.

Överby A, Pettersson R, Grünewald K, Huiskonen JT (2008) Insights into Bunyavirus architecture from electron cryo-tomography of Uukuniemi virus. PNAS 105: 2375-2379.

2007

Schelhaas M, Pelkmans L, Malmström J, Aebersold R, Haugstetter J, Ellgaard L, Grünewald K, Helenius A (2007) Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells. Cell 131: 516-529.

2006

Grünewald K, Cyrklaff M (2006) Structure of complex viruses and virusinfected cells by electron cryo tomography. Curr. Opin. Microbiol. 9: 437-442.

Zanetti G, Briggs JA, Grünewald K, Sattentau QJ, Fuller SD (2006) Cryoelectron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathog. 2: e83.

Briggs AG, Grünewald K, Glass B, Förster F, Kräusslich HG, Fuller SD (2006) The mechanism of HIV-1 core assembly: insights from threedimensional reconstructions of authentic virions. Structure 14: 15-20.

2005

Cardone G, Grünewald K, Steven AC (2005) A resolution criterion for electron tomography based on cross-validation. J. Struct. Biol. 151: 117-129.

2003

Grünewald K, Desai P, Winkler DC, Heymann JB, Belnap DM, Baumeister W, Steven AC (2003) Three-dimensional structure of Herpes simplex virus from cryo-electron tomography, Science 302: 1396-1398.54

Grünewald K, Medalia O, Gross A, Steven A C, Baumeister W (2003) Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: Implications of crowding. Biophysical Chem. 100: 577-591.

2001

Grünewald K, Hirschberg J, Hagen C (2001) Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis. J. Biol. Chem. 276: 6023-6029.

Grünewald K, Hagen C (2001) ß-carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis (Chlorophyceae). J. Appl. Phycol. 13: 89-93.

Hagen C, Grünewald K, Xyländer M, Rothe E (2001) Effect of cultivation parameters on growth and pigment biosynthesis in flagellated cells of Haematococcus pluvialis. J. Appl. Phycol. 13: 79-87.

2000

Hagen C, Grünewald K, Schmidt S, Müller J (2000) Accumulation of secondary carotenoids in flagellates of Haematococcus pluvialis (Chlorophyta) is accompanied by an increase in per unit chlorophyll productivity of photosynthesis. Eur. J. Phycol. 35: 75-82.

Grünewald K, Hagen C (2000) Extrusion of secondary carotenoid containing vesicles from flagellates of Haematococcus pluvialis. (Volvocales, Chlorophyceae) J. Appl. Bot. 74: 141-144.

Hagen C, Grünewald K (2000) Fosmidomycin as an inhibitor of the nonmevalonate terpenoid pathway depresses synthesis of secondary carotenoids in flagellates of the green alga Haematococcus pluvialis. J. Appl. Bot. 74: 137-140.

Grünewald K, Eckert M, Hirschberg J, Hagen C (2000) Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae). Plant Physiol. 122: 1261-1268.

1997

Grünewald K, Hagen C, Braune W (1997) Secondary carotenoid accumulation in flagellates of the green alga Haematococcus lacustris. Eur. J. Phycol. 32: 387- 392.

1994

Grünewald K (1994) Microsphaera syringae mit Cleistothecien. Boletus 18:25-26.