Technology NetworkHDcryoNet

The Cryo-Electron Microscopy Network of Heidelberg University

The Cryo-Electron Microscopy Network of Heidelberg University (HDcryoNet) hosts cryo-EM infrastructure located at the BioQuant and at the Biochemistry Center. The aim of HDcryoNet is to foster collaborative research relying on cryo-EM methods, and to teach these methods to students at Heidelberg University. HDcryoNet operates as a network of users who independently use the equipment and share duties. HDcryoNet operates four high-end cryo-EM instruments.

Logo-illustration: the writing HDcrynoNet in white letters is positioned above three gray oval grids. Behind this, there is a vertical yellow light ray, and at its top a black „e-“ is to be seen. Five squares are positioned in a half-circle at the bottom of the illustration, functioning as a foundation of the logo.

Instruments located at the BioQuant

Aquilos cryo-FIB/SEM

The Aquilos dual-beam microscope is a dedicated instrument for the preparation of thin cryo-lamellae of vitrified cells that are subsequently studied by cryo-electron tomography in the Krios cryo-TEM. The microscope is equipped with a focused Gallium ion beam column, an electron beam column, and a cryo-stage. It uses an Edward-Thornley detector and in lens and in column Trinity detection systems for detection of secondary and backscattered electrons. The microscope can be operated in a semi-automated fashion using Autoscript scripting. The microscope is located in the BSL2 suite together with the Krios microscope to support in situ cryo-electron tomography projects. The microscope was acquired through DFG funding and is in operation since 2018.

Building: INF 229
Room: U42&45 (Biosafety level 2)
Phone: +49 6221 5419373

 

Content image: Photograph of the Aquilos dual-beam microscope

Krios – cryo-TEM (300kV)

The Krios cryo-transmission electron microscope is usually operated at 300kV and is dedicated to cryo-electron tomography and high-resolution data collection for single-particle analysis. It is equipped with a field emission gun, K3 Direct Electron Detector, Quanta GIF imaging filter, Volta phase plate and a dual-axis stage. Recently it has been upgraded with a state-of-the-art 3rd generation Autoloader.  Data collection is carried out remotely using EPU or SerialEM. The microscope is located in the BSL2 suite together with the Aquilos microscope. The microscope was acquired through CellNetworks funding and is in operation since 2012.

Building: INF 229
Room: U41 (Biosafety Level 2)
Phone: +49 6221 5419370

 

Content Image: A 3D-image of the Krios cryo-transmission electron microscope

Instruments located at BZH

Glacios – cryo-TEM (200kV)

The Glacios is a state-of-the-art 200kV cryo-transmission electron microscope equipped with an Autoloader that allows streamlined sample screening and data collection for single-particle analysis of purified complexes. It is equipped with a field emission gun, Falcon3-EC direct detector, and a Volta phase plate. Data collection is carried out remotely using EPU or SerialEM. The microscope is located in vicinity of the Talos L120C microscope to support single-particle projects. The microscope was acquired through DFG funding and is in operation since 2020.

Building: INF 328
Phone: +49 6221 544377

 

Content Image: A photograph of the Glacios 200kV cryo-transmission electron microscope

Talos L120C – (cryo)-TEM 120 kV

Talos L120C is a state-of-the-art 120kV transmission electron microscope that can be operated at cryogenic temperatures. It is equipped with a LaB6 electron source and a Ceta-M camera with a 4k × 4k CMOS sensor. This highly versatile microscope is dedicated to sample screening at both room and cryogenic temperatures. It operates with side-entry holders (several holders are available, incl. the newest Elsa Cryo-Transfer Holder). The microscope was acquired through DFG funding and is in operation since 2019.

Building: INF 328
Phone: +49 6221 544165

 

Content image: Photograph of the Talos L120C 120kV transmission electron microscope

Cryo-EM sample preparation instruments

Automatic plunge freezers:
Vitrobot Mark IV (BioQuant), Vitrobot Mark IV (BZH), Gatan Cryoplunge 3 (BioQuant), Leica GP2 (CIID)

Glow dischargers:
Gatan Solarus 950 (BioQuant)

Data storage and processing infrastructure

The computing center of Heidelberg University provides access to a fast large scale data storage facility (Scientific Data Storage) and a high-performance computing cluster (bwForCluster) equipped with multi-GPU nodes tailored for cryo-EM data analysis.

More information on the high-performance computing cluster (bwForCluster):

hpc-support@urz.uni-heidelberg.de

Further information and training related to computational resources available on campus at Heidelberg University are provided by the Community Forum Data-Intensive Computing and the HPC-Support.

STEERING COMMITTEE

Current members of the HDcryoNet steering committee (ordered alphabetically):

- Petr Chlanda (BioQuant/Department of Infectious Diseases-Virology)

- Dirk Flemming (BZH)

- Wim Hagen (EMBL Heidelberg)

- Stefan Pfeffer (ZMBH)

- Rasmus R. Schröder (BioQuant)

- Irmi Sinning (BZH)

CONTACT

CRYO-EM Gallery

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    Content image: 3 Microscopic images of cells infected with Sars-Cov-2
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    Content image: Microscopic image of yellow, red and green highlighted cell membrane sheets
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    Content image: an illustration, possibly of a virus. On a gray background one can see spiraling strings in turquoise, blue and orange, partly intertwined, their top parts reaching into a horizontally separated area at the top of the picture.
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    Content image: Microscopic image of Ebola-virus-like particles.
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    Content image: Microscopic colored image of a vertebrate gamma tubulin ring complex
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    Content image: Microscopic colored image of a fungal gamma-tubulin small complex
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    Content image: Microscopic partly colored image of a Human Ebp1-ribosome complex
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    Content image: Microscopic partly colored image of a bacterial ribosome-associated quality control complex

RECENT PUBLICATIONS

2021

Soni K, Kempf G, Manalastas-Cantos K, Hendricks A, Flemming D, Guizetti J, Simon B, Frischknecht F, Svergun DI, Wild K, Sinning I., 2021. Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation. Commun Biol. 4(1):600, doi: 10.1038/s42003-021-02132-y.

 

Lau B, Cheng J, Flemming D, La Venuta G, Berninghausen O, Beckmann R, Hurt E., 2021. Structure of the Maturing 90S Pre-ribosome in Association with the RNA Exosome. Mol Cell 81, 283-303. doi: 10.1016/j.molcel.2020.11.009

 

Filbeck, S.*, Cerullo, F.*, Paternoga, H., Tsaprailis, G., Joazeiro, C.A.P.#, Pfeffer, S#, 2021. Mimicry of canonical translation elongation underlies alanine tail synthesis in RQC. Mol Cell 81, 104-114.

 

Winter SL, Chlanda P. Dual-axis Volta phase plate cryo-electron tomography of Ebola virus-like particles reveals actin-VP40 interactions, J Struct Biol., 2021, doi: 10.1016/j.jsb.2021.107742

 

Klein S, Wachsmuth-Melm M, Winter SL, Kolovou A, Chlanda P. Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells, Methods in Cell Biology, 2021, doi.org/10.1016/bs.mcb.2020.12.009

 

Klein S*, Wimmer BH*, Winter SL, Kolovou A, Laketa V, Chlanda P (2021), Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies. Commun Biol. 2021 4(1):137. doi: 10.1038/s42003-020-01567-z.

 

Würtz, M., Böhler, A., Neuner, A., Zupa, E., Rohland, L., Liu, P., Vermeulen, B.J.A, Pfeffer, S., Eustermann, S., Schiebel, E., 2021. Reconstitution of the recombinant human gamma-tubulin ring complex. Open Biol 11: 200325

 

2020

Zupa, E.*, Zheng, A.*, Neuner, A., Würtz, M., Liu, P., Böhler, A., Schiebel, E.#, Pfeffer, S#, 2020. The cryo-EM structure of a gamma-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems. Nat Commun 11, 5705.

 

McDowell MA, Heimes M, Fiorentino F, Mehmood S, Farkas Á, Coy-Vergara J, Wu D, Bolla JR, Schmid V, Heinze R, Wild K, Flemming D, Pfeffer S, Schwappach B, Robinson CV, Sinning I. Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex. Mol Cell. 2020 Oct 1;80(1):72-86.e7. doi: 10.1016/j.molcel.2020.08.012. Epub 2020 Sep 9. PMID: 32910895.

 

Wild K, Aleksić M, Lapouge K, Juaire KD, Flemming D, Pfeffer S, Sinning I. MetAP-like Ebp1 occupies the human ribosomal tunnel exit and recruits flexible rRNA expansion segments. Nat Commun. 2020 Feb 7;11(1):776. doi:

10.1038/s41467-020-14603-7. PMID: 32034140; PMCID: PMC7005732.

 

Klein S*, Cortese M*, Winter SL*, Wachsmuth-Melm M, Neufeldt CJ, Cerikan B, Stanifer ML, Boulant S, Bartenschlager R#, Chlanda P#. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. Nat Commun. 2020 Nov 18;11(1):5885. doi: 10.1038/s41467-020-19619-7

 

2019

Liu P, Zupa E, Neuner A, Böhler A, Loerke J, Flemming D, Ruppert T, Rudack T, Peter C, Spahn C, Gruss OJ, Pfeffer S, Schiebel E. Insights into the assembly and activation of the microtubule nucleator γ-TuRC. Nature. 2020 Feb;578(7795):467-471. doi: 10.1038/s41586-019-1896-6. Epub 2019 Dec 19. PMID:31856152