Technology Platform Electron Cryo-Microscopy

We enable scientists who wish to use cryo-EM in their own research portfolio to become independent investigators in our facility. To this end, we have developed practical and theoretical courses for data acquisition and data analysis at different levels. We also support technology projects that create synergies between the electron cryo-microscopy platform and the Science Campus Bahrenfeld and are supported by third-party funds.

Our Cryo-EM team is pleased to offer researchers the opportunities to make a valuable contribution to their research projects through the use of cryo-electron microscopy. The team of the Cryo-Electron Microscopy Technology Platform is happy to support you in planning and performing experiments and in data analysis.

For further questions, contact cryoem"AT"cssb-hamburg.de.

10 to 21 February 2025
Basic Principles and Practical Aspects of Cryo-EM SPA and Tomography (Module 62-485.1)

2023

Ferreira JL, Prazak V, Vasishtan D, Siggel M, Hentzschel F, Binder AM, Pietsch E, Kosinski J, Frischknecht F, Gilberger TW, Grunewald K (2023) Variable microtubule architecture in the malaria parasite. Nat Commun 14: 1216 doi: 10.1038/s41467-023-36627-5

Seyfert CE, Porten C, Yuan B, Deckarm S, Panter F, Bader CD, Coetzee J, Deschner F, Tehrani KHME, Higgins PG, Seifert H, Marlovits TC, Herrmann J, Muller R (2023) Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering. Angew Chem Int Edit 6210.1002/anie.202214094

Williams HM, Thorkelsson SR, Vogel D, Milewski M, Busch C, Cusack S, Grunewald K, Quemin ERJ, Rosenthal M (2023) Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein. Nucleic Acids Research 51: 1424-1442 doi: 10.1093/nar/gkac1249

2022

Bucker R, Seuring C, Cazey C, Veith K, Garcia-Alai M, Grunewald K, Landau M (2022) The Cryo-EM structures of two amphibian antimicrobial cross-beta amyloid fibrils. Nature Communications 13ARTN 4356 10.1038/s41467-022-32039-z

Franken LE, Rosch R, Laugks U, Grunewald K (2022) Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells. STAR Protoc 3: 101696 doi: 10.1016/j.xpro.2022.101696

Killer M, Finocchio G, Mertens HDT, Svergun DI, Pardon E, Steyaert J, Loew C (2022) Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C. Front Mol Biosci 9ARTN 917725 10.3389/fmolb.2022.917725

Niebling S, Veith K, Vollmer B, Lizarrondo J, Burastero O, Schiller J, Garcia AS, Lewe P, Seuring C, Witt S, Garcia-Alai M (2022) Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins. Front Mol Biosci 9ARTN 882288 10.3389/fmolb.2022.882288

Pazicky S, Alder A, Mertens H, Svergun DI, Gilberger T, Low C (2022) N-terminal phosphorylation regulates the activity of Glycogen Synthase Kinase 3 from Plasmodium falciparum. Biochem J 10.1042/BCJ20210829

Villalta A, Schmitt A, Estrozi LF, Quemin ER, Alempic JM, Lartigue A, Prazak V, Belmudes L, Vasishtanm D, Colmant AM, Honore FA, Coute Y, Grunewald K, Abergel C (2022) The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield. Elife 11ARTN e77607 10.7554/eLife.77607

Wald J, Fahrenkamp D, Goessweiner-Mohr N, Lugmayr W, Ciccarelli L, Vesper O, Marlovits TC (2022) Mechanism of AAA plus ATPase-mediated RuvAB-Holliday junction branch migration. Nature 609: 630-+ doi: 10.1038/s41586-022-05121-1

2021

Albers S, Beckert B, Matthies MC, Mandava CS, Schuster R, Seuring C, Riedner M, Sanyal S, Torda AE, Wilson DN, Ignatova Z (2021) Repurposing tRNAs for nonsense suppression. Nature Communications 1210.1038/s41467-021-24076-x

Ayyer K, Xavier PL, Bielecki J, Shen Z, Daurer BJ, Samanta AK, Awel S, Bean R, Barty A, Bergemann M, Ekeberg T, Estillore AD, Fangohr H, Giewekemeyer K, Hunter MS, Karnevskiy M, Kirian RA, Kirkwood H, Kim Y, Koliyadu J, Lange H, Letrun R, Lubke J, Michelat T, Morgan AJ, Roth N, Sato T, Sikorski M, Schulz F, Spence JCH, Vagovic P, Wollweber T, Worbs L, Yefanov O, Zhuang YL, Maia FRNC, Horke DA, Kupper J, Loh ND, Mancuso AP, Chapman HN (2021) 3D diffractive imaging of nanoparticle ensembles using an x-ray laser. Optica 8: 15-23 doi: 10.1364/Optica.410851

Bunduc CM, Fahrenkamp D, Wald J, Ummels R, Bitter W, Houben ENG, Marlovits TC (2021) Structure and dynamics of a mycobacterial type VII secretion system. Nature 593: 445-448 doi: 10.1038/s41586-021-03517-z

Killer M, Wald J, Pieprzyk J, Marlovits TC, Low C (2021) Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes. Sci Adv 7: eabk3259 doi: 10.1126/sciadv.abk3259

Kotov V, Lunelli M, Wald J, Kolbe M, Marlovits TC (2021) Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies. Biochem Biophys Rep 27: 101039 doi: 10.1016/j.bbrep.2021.101039

Kotov V, Mlynek G, Vesper O, Pletzer M, Wald J, Teixeira-Duarte CM, Celia H, Garcia-Alai M, Nussberger S, Buchanan SK, Morais-Cabral JH, Loew C, Djinovic-Carugo K, Marlovits TC (2021) In-depth interrogation of protein thermal unfolding data with MoltenProt. Protein Sci 30: 201-217 doi: 10.1002/pro.3986

Kouba T, Vogel D, Thorkelsson SR, Quemin ERJ, Williams HM, Milewski M, Busch C, Gunther S, Grunewald K, Rosenthal M, Cusack S (2021) Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity. Nat Commun 12: 7018 doi: 10.1038/s41467-021-27305-5

Miletic S, Fahrenkamp D, Goessweiner-Mohr N, Wald J, Pantel M, Vesper O, Kotov V, Marlovits TC (2021) Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation. Nat Commun 12: 1546 doi: 10.1038/s41467-021-21143-1

Pfitzner S, Bosse JB, Hofmann-Sieber H, Flomm F, Reimer R, Dobner T, Gruenewald K, Franken LE (2021) Human Adenovirus Type 5 Infection Leads to Nuclear Envelope Destabilization and Membrane Permeability Independently of Adenovirus Death Protein. International Journal of Molecular Sciences 22ARTN 13034 10.3390/ijms222313034

Prazak V, Grunewald K, Kaufmann R (2021) Correlative super-resolution fluorescence and electron cryo-microscopy based on cryo-SOFI. Methods Cell Biol 162: 253-271 doi: 10.1016/bs.mcb.2020.10.021

Silvester E, Vollmer B, Prazak V, Vasishtan D, Machala EA, Whittle C, Black S, Bath J, Turberfield AJ, Grunewald K, Baker LA (2021) DNA origami signposts for identifying proteins on cell membranes by electron cryotomography. Cell 184: 1110-1121 e1116 doi: 10.1016/j.cell.2021.01.033

Yuan B, Portaliou AG, Parakra R, Smit JH, Wald J, Li YC, Srinivasu B, Loos MS, Dhupar HS, Fahrenkamp D, Kalodimos CG, van Hoa FD, Cordes T, Karamanou S, Marlovits TC, Economou A (2021) Structural Dynamics of the Functional Nonameric Type III Translocase Export Gate. Journal of Molecular Biology 433ARTN 167188 10.1016/j.jmb.2021.167188

2020

Franken LE, Grunewald K, Boekema EJ, Stuart MCA (2020) A Technical Introduction to Transmission Electron Microscopy for Soft-Matter: Imaging, Possibilities, Choices, and Technical Developments. Small: e1906198 doi: 10.1002/smll.201906198

Lunelli M, Kamprad A, Burger J, Mielke T, Spahn CMT, Kolbe M (2020) Cryo-EM structure of the Shigella type III needle complex. PLoS Pathog 16: e1008263 doi: 10.1371/journal.ppat.1008263

Quemin ERJ, Machala EA, Vollmer B, Prazak V, Vasishtan D, Rosch R, Grange M, Franken LE, Baker LA, Grunewald K (2020) Cellular Electron Cryo-Tomography to Study Virus-Host Interactions. Annu Rev Virol 7: 239-262 doi: 10.1146/annurev-virology-021920-115935

Vogel D, Thorkelsson SR, Quemin ERJ, Meier K, Kouba T, Gogrefe N, Busch C, Reindl S, Gunther S, Cusack S, Grunewald K, Rosenthal M (2020) Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein. Nucleic Acids Res 48: 5749-5765 doi: 10.1093/nar/gkaa253

Vollmer B, Prazak V, Vasishtan D, Jefferys EE, Hernandez-Duran A, Vallbracht M, Klupp BG, Mettenleiter TC, Backovic M, Rey FA, Topf M, Grunewald K (2020) The prefusion structure of herpes simplex virus glycoprotein B. Sci Adv 610.1126/sciadv.abc1726

Wolff G, Limpens R, Zevenhoven-Dobbe JC, Laugks U, Zheng S, de Jong AWM, Koning RI, Agard DA, Grunewald K, Koster AJ, Snijder EJ, Barcena M (2020) A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369: 1395-1398 doi: 10.1126/science.abd3629

2019

Moser F, Prazak V, Mordhorst V, Andrade DM, Baker LA, Hagen C, Grunewald K, Kaufmann R (2019) Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy. Proc Natl Acad Sci U S A 116: 4804-4809 doi: 10.1073/pnas.1810690116

Nikolaus Goessweiner-Mohr VK, Matthias J. Brunner, Julia Mayr, Jiri Wald, Lucas Kuhlen, Sean Miletic, Oliver Vesper, Wolfgang Lugmayr Samuel Wagner, Frank DiMaio, Susan Lea, Thomas C., Marlovits (2019) Structural control for the coordinated assembly into functional pathogenic type-3 secretion systems. BioArxiv https://doi.org/10.1101/714097

The cryo-EM technology platform provides access to the following microscopes, all of which can be operated via remote access.

Talos L120c

Thermo Fisher Scientific, 120 kV accelerating voltage and LaB₆ thermionic source

This is an entry level screening microscope with a CETA camera, mainly for negative stain samples, but also capable of cryo-EM using side-entry holders from Gatan. This microscope is used for training purposes and evaluation of the first steps of primarily single particle sample preparation. It is further equipped with a LAMBDA next-generation pixel detector capable to acquire electron diffraction data. Software options include SerialEM and Velox.

Talos Arctica

Thermo Fisher Scientific, 200 kV accelerating voltage and field emission gun (X-FEG)

It is equipped with an autoloader, Falcon IVi direct detector and phase plate. This microscope is used primarily for cryo-EM sample optimization and single particle analysis. Software options include Thermo Fisher based Software solutions and SerialEM.

Titan Krios G3

Thermo Fisher Scientific, 300 kV accelerating voltage and field emission gun (X-FEG) source

Top-end microscope with fringe-free imaging, autoloader, Bioquantum energy filter, K3 direct detector, phase plate and dual axis tilt stage. This microscope is used for cryo-EM single particle and tomography data acquisition. Software options include Thermo Fisher based Software solutions, and SerialEM.

Titan Krios G3i

Thermo Fisher Scientific, 300 kV accelerating voltage and field emission gun (X-FEG) source

Top-end microscope with fringe-free imaging, autoloader, Bioquantum energy filter, K3 direct detector, phase plate. This microscope is used for cryo-EM single particle and tomography data acquisition.  Software options include Thermo Fisher based Software solutions, and SerialEM.

Aquilos 2 Cryo-FIB/SEM

Thermo Fisher Scientific

Cryo-DualBeam system with focused ion beam and scanning electron microscope (FIB-SEM) dedicated to the preparation of thin lamellae from primarily vitreous cells for subsequent electron cryo-tomography. Recent upgrades include iFLM and cryo-lift out. The Aquilos is able to perform automated milling and software options include xT and Thermo Fisher Software packages.

Arctis Cryo-Plasma FIB/SEM

Thermo Fisher Scientific

Arctis Cryo-Plasma FIB from Thermo Fisher Scientific, highly automated Plasma FIB system with three different gas sources (xenon, oxygen, argon), in-line fluorescence microscope (iFLM) for easy milling targeting and 12 grid autoloading system, allowing seamless transition to cryo-TEM. The Arctis is used for preparation of high-quality thin lamellae from vitreous cells as well as high pressure frozen waffle samples. The available software specifically developed for fully automated milling is based on the web-based interface WebUI for remote access. The more conventional correlation and milling software Maps and AutoTEM are available.

Equipment for specimen preparation and mammalian cell culture

Users have access to a broad range of auxiliary equipment and dedicated laboratory space for the last steps of specimen preparation and mammalian cell culture, right before their vitrification.