The Multi-User Cryo-EM facility operated by the University of Hamburg is located within the Centre of Structural Systems Biology (CSSB) on the Science Campus Bahrenfeld, Hamburg, Germany. We support a variety of structural biology projects with a focus on host-pathogen interactions.
The facility has established efficient workflows primarily for the modalities of cryo-EM, i.e. cryo-electron tomography (cryo-ET) and single particle analysis (SPA). The cryo-CLEM technique is being arranged through our close interaction with the CSSB Advanced Light and Fluorescence Microscopy (ALFM) facility. Independent usage and collaborative research projects with research groups are possible.
Our mission:
To provide access to state-of-the-art cryo-EM instruments for data collection towards atomic resolution structure determination of biochemically purified single particles and for the study of cellular structures.
To enable scientists who want to use cryo-EM in their own research portfolio to become independent investigators.
To support method development projects that generate links between facilities and infrastructures on campus.
The facility space has various preparation devices and lab space for cryo-specimen preparation (particles and cells), data quality assessment tools for ‘’on-the-fly’’ evaluation and visualization, and image data storage capacity on a short-term basis. We provide expert staff that trains, assists and advises users at different levels, both on-site and remotely. Our staff is available for consultations beforehand and in a follow-up.
Our facility is available to researchers from all CSSB partner institutions, as well as external academic users. Collaborative data collection involving facility staff of pre-screened samples can be negotiated on a case-by-case basis.
The Multi-User cryo-EM Facility at CSSB was established with substantial support from the DFG.
technical support for microscopes for state-of-the-art data acquisition
support in the experimental design and getting started with cryoEM
a well-maintained computing environment and guidance for initial data analysis
provision of various data processing pipelines
mutual development of user-specific workflows (both for data acquisition and processing)
confluence space (extended information, guidelines, training manuals)
regular user meetings, mailing list, confluence space with extended information
yearly cryo-EM workshops
Instrumentation
Electron Microscopes
Talos L120C from Thermo Fisher Scientific, 120 kV accelerating voltage and LaB6 thermionic source. This is a robust 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 sample preparation. SerialEM is installed.
Talos Arctica from Thermo Fisher Scientific, 200 kV accelerating voltage and field emission gun (X-FEG) source. It is equipped with an autoloader, Falcon 3EC direct detector and phase plates. This microscope is used primarily for Cryo-EM sample optimization, single particle analysis and electron diffraction. SerialEM is installed. The microscope is maneuverable by remote.
Titan Krios G3 from Thermo Fisher Scientific, 300 kV accelerating voltage and field emission gun (X-FEG) source. Top-end microscope with 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. SerialEM is installed. The microscope is maneuverable by remote.
Titan Krios G3i from Thermo Fisher Scientific, 300 keV accelerating voltage and field emission gun (X-FEG) source. Top-end microscope with autoloader, Bioquantum energy filter, Falcon 3EC (pre-filter) and K3 direct detector, phase plate and single axis tilt stage. This microscope is used for Cryo-EM single particle and tomography data acquisition. SerialEM is installed. The microscope is maneuverable by remote.
Aquilos 2 Cryo-FIB from 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. Automilling implemented. Remote operation planned.
Users preparing biological smples for examination with our high-end microscope (Titan Krios II) IMAGE: Jörg Müller
Training on the Talos L120. IMAGE: Jörg Müller
Talos Arctica - Sample screening is an initial step prior to use of the high-end microscopes. IMAGE: Andrea Impagniatiello
Aquilos 2 FIB-SEM - Preparing cells for tomography. IMAGE: Andrea Impagniatiello
Sample Preparation and Auxillary Equipment
Users have access to a broad range of auxiliary equipment and dedicated laboratory space for specimen preparation and mammalian cell culture.
two glow discharger (Quorum GloQube & Harrick Plasma Cleaner PDC-002-CE)
Vitrobots Mark IV and MPI Martinsried type manual plunge freezer for BSL-1 work
Vitrobots Mark IV and Leica GP2 plunge freezers for BSL-2 work, housed in custom-designed Berner Safety Cabinets
Leica ACE60 carbon/gold/platinum coater
dedicated laboratory space for specimen preparation and mammalian cell culture, including:
CO2 incubators and Light Microscopes (DMIL and DMi1 from Leica, SteREO Discovery, V8 from Zeiss)
two safety hoods (Berner)
two incubators (Heracell VIOS 250i CO2, Thermo Fisher Scientific)
Safety cabinet berner, IMAGE: Jörg Müller
Quorum GloQube
Harrick Plasma Cleaner PDC-002
Thermo Vitrobots Mark IV
Leica GP2
Leica ACE
Safety Cabinets
CO2 incubators
Zeiss Stereo microscope
Leica DMi1
Leica light microscope
Cryo-EM holder
The Multi-User CryoEM Facility is located on DESY Campus, Notkestr. 85, Building 15.For instructions on travelling to CSSB, see here (link internal CSSB).
Authorised access only. Visitors to the main Laboratory Area must be under the supervision of a Cryo-EM staff member or an authorised User at all times.
Booking equipment
Internal users can make instrument bookings through the PPMS booking system once they have completed training. To request booking onto a training course or to find out more information email cryoem@cssb-hamburg.de
Costs
Different costs apply for internal and external groups. Costs comply with the DFG guidelines. The responsibility to ensure funding lies with the user group.
How to get Access as a New User or Group
Access costs comply with the DFG guidelines and apply for all usage modalities. Internal users can make instrument bookings through the PPMS booking system once they have completed training. To book a training course or receive more information about the facility please send an e-mail to: cryoem@cssb-hamburg.de
Access for new users or groups
Before starting a new project, please e-mail carolin.seuring@cssb-hamburg.de with your request for access and to schedule an initial consultation. Please provide the following information:
Name, group, affiliation
Type of project, timeline, funding, preliminary data etc.
independent usage, collaboration with CSSB group or training
your experience level in cryoEM (equipment used, software, etc.)
whether you are planning BSL-1 or BSL-2 experiments
Note: Additional procedures apply to register pathogens associated with BSL-2 projects
Visit our Confluence Spaceor request confluence access (if non-CSSB and non-DESY), download and sign the usage rules and etiquette
Attend the new user orientation meeting, held monthly.
Location: Zoom
Bring signed Usage Rules, Etiquette
Equipment
Modes of usage
Training and Observation Time
List of EM supplies needed
EM user mailing list subscriptions
Virtual Tour/Demo
Computing and data transfer resources
PPMS and how to schedule Training
Safety Trainings needed to proceed
When completed, provide copies of Safety Trainings
You must complete all safety trainings before you can work in the facility.
Create an account in PPMS, project request and financial account information
Schedule training
Attend scheduled training and proceed to facility use
You will also need to:
in case of BSL2 work help to update hygiene plan, disposal plan and onsite SOPs
create accounts for extended Confluence access and for access to Beegfs (Transfer)
subscribe to em-user mailing list to be informed about newest developments
Training on all instruments is mandatory to ensure functional safe operation of our highly specialized equipment. Separate training is necessary for every instrument including each microscope. Our training modules aim to provide users with the necessary skills to acquire electron microscopy data. Users will be graded according to their level of experience for each respective microscope. Records are kept with the Facility Staff.
Training Modules
1. Negative stain and sample preparation training
Description: Training on general negative stain electron microscopy sample preparation. Including theory, handling of grids, glow discharging (GlowQube, Harrick Plasma Cleaner) Prerequisites: New User Orientation (see Chapter 2 - New User Access Procedure) Duration: typically 2-hours session with EM facility staff, dependent on requirements and aptitude Occupancy: Maximum 2 people Supplies provided by facility: Negative stain reagents depending on requirement, filter paper Supplies to be provided by users: Prepared sample, grid box, continuous carbon 300 mesh grids, tweezers
2. Carbon Coater
Description: Training on using the Leica ACE600 carbon coater, practical application, recipe creation Prerequisites: New User Orientation, Negative stain and sample preparation training Duration: typically 1-hour session with EM facility staff Occupancy: Maximum 2 people Supplies provided by facility: Carbon rods, Mica Supplies to be provided by users: Formvar coated or other grids
3. Vitrobot Plunge freezer
Description: Training on using the Vitrobot plunge freezer, cryo-sample preparation theory, setup, practical application, use of Harrick plasma cleaner (or GloQube) Prerequisites: New User Orientation Duration: up to 2 separate, 2-hour sessions with EM facility staff member Occupancy: Maximum 1 person Supplies provided by facility: plunging tweezers Supplies to be provided by users: Whatman Paper Grade 1 Ø 55mm, cryo-optimised sample, cryo-grid box with lid, cryo-grids according to user requirements
4. Manual Plunge freezer
Description: Training on using the manual plunge freezer. Prerequisites: New User Orientation Duration: up to 2 separate, 2-hour sessions with EM facility staff, 1 hour if prior training on cryo-plungers was completed Occupancy: Maximum 1 person Supplies provided by facility: Filter paper (Whatman #1), plunging tweezers (for training only) Supplies to be provided by users: cryo-optimised sample, cryo-grid box with lid, cryo-grids according to user requirements, plunging tweezers (after training)
5. Leica GP2 Plunge freezer
Description: Training on using the Leica GP2 Plunge freezer. Prerequisites: New User Orientation Duration: up to 2 separate, 2-hour sessions with EM facility staff, 1 hour if prior training on cryo plungers was completed Occupancy: Maximum 1 person Supplies provided by facility: plunging tweezers Supplies to be provided by users: Whatman Paper Grade 1 Ø 55mm, cryo-optimised sample, cryo-grid box with lid, cryo-grids according to user requirements
6. Autogrid Clipping
Description: Training on preparing cryoEM Autogrids Prerequisites: New User Orientation, negative stain and sample preparation training Duration: typically around 2-hour session, 1 hour demonstration by EM facility staff and 1 hour practice Occupancy: Maximum 1 person Supplies provided by facility: Clipping tools, short term storage for cryo-grids (max 2 weeks) Supplies to be provided by users: Autogrid cryo-box, plunged cryoEM grids, autogrid rings and C-clips
7. Talos L120
Description: Training and hands-on practical safe use of the scope for screening negative stain grids. Theory, microscope hardware, loading samples, aligning the scope, imaging strategies, focusing, aberrations, troubleshooting. Standard known sample provided or own sample. Prerequisites: New User Orientation, negative Stain and sample preparation training Duration: up to 2 separate, 2-hour sessions with EM facility staff, more if necessary Observation session with independent user: 4 hours Final approvaland sign off after 10-20 hours of supervised instrument time logged and demonstration of proficiency Occupancy: 1-2 people
8. Talos Arctica and Titan Krios
Description: Training and hands-on practical use of Arctica and Krios for screening cryo-grids and setting up high-resolution energy filtered data collection (npEFTEM) with K3 (EPU and Tomo). Theory, microscope hardware, aligning the scope, imaging strategies, focusing, aberrations, troubleshooting. Prerequisites: negative stain and sample preparation training, Talos L120 independence, plunge freezing and Autogrid clipping proficiency Duration: up to 2 separate 4-hour sessions with EM facility staff, more if necessary Observation session with independent user: 2 x 4 hours Final approval and sign off after ~100 hours of supervised instrument time logged by facility staff and demonstration of proficiency Occupancy: 1-2 people Supplies needed: screened and vitrified autogrids with proven quality
9. Tomography
Description: theory and hands-on training of tomography data acquisition in batch with TFS Tomography software and/or serialEM at the Krios microscopes. Includes specific alignment steps and dose calibration of the K3 direct detector for tilt series acquisition. Before introduction to serialEM, principles of tomography software should be understood with the more basic TFS Tomo. Prerequisites: sample preparation training, plunge freezing and Autogrid clipping proficiency, introduction to the electron microscope alignment at the Talos L120, introduction to the Krios with energy filter and K3 direct detector Duration: up to 2 separate 8-hour sessions with EM facility staff, more if necessary Observation session with independent user: 2 x 4 hours, probably more will be needed Final approval and sign off after ~100 hours of supervised instrument time logged by facility staff and demonstration of proficiency Occupancy: 1-2 people Supplies needed: Vitrified autogrids
10. Aquilos FIB SEM
Description: theory and hands-on training of preparing FIB-thinned lamellae from cellular samples including preparation steps for cryo work on the scope. The microscope software XTUI and the additional softwares MAPS and AutoLamella for autonomous lamella preparation will be trained. Prerequisites: sample preparation training of cellular samples, plunge freezing and Autogrid clipping proficiency. Duration: up to 2 separate 8-hour sessions with EM facility staff, more if necessary Observation session with independent user: 2 x 4 hours Final approval and sign off after ~100 hours of supervised instrument time logged by facility staff and demonstration of proficiency Occupancy: 1-2 people Supplies needed: Vitrified FIB-milling autogrids
In order to acquire proficiency on any instrument, users are encouraged to join experienced user sessions. This allows new users to see different imaging strategies specific to their requirements.
Joint Trainings
Joint trainings complement individual training modules and will allow new users with similar experiences and requirements to spend time together on the microscope, exchange knowledge and foster learning.
Additional Training
After you have completed your initial training, observation time and group training - you may require additional training. We also offer refresher training to ensure that you know how to correctly operate the equipment, and inform you of any recent updates in operating procedures. For additional trainings, please contact cryoem@cssb-hamburg.de, to discuss further needs.
CryoEM Learning
To get started in CryoEM, we recommend to look into the following excellent online resources:
We recommend to subscribe to these mailing lists:
3DEM email list - community news, technical discussion, data processing, job advertisement
CCPEM email list - single particle data acquisition and processing
SerialEM Discussion group - troubleshooting feedback by the developers
Jensen’s course together with a practical video tutorial on SPA by Thermo Fisher Scientific and the NIH, the em-learning platform has collected all the knowledge in one place. It is free, but registration is required
Lecture series by the SBGrid consortium on relevant software packages for data processing
The National Resource for Automated Molecular Microscopy (NRAMM) offers a helpful list of Lectures from workshops
Workshops on acquisition and processing software can be found here:
The facility collaborates with users on scientific projects with a particular focus on method development, and for workflow validation and extension. We particularly aim to combine technologies on campus and develop new methods between CSSB facilities and with different technologies on campus (Petra III, XFEL, sample delivery).
"Plasmofraction"
This project will use a bottom-up structural systems biology approach to investigate membrane protein complexes in the transmissible gametocyte stages of the malaria parasite Plasmodium falciparum.
Collaborators: Filarski, Kosinski, Löw, Witt, Seuring
Funding: CSSB Flagship Project
"Jetfreezing - Novel Sample Delivery Approaches for CryoEM"
In this project we will combine XFEL sample delivery methods with cryoEM imaging.
Collaborators: Bajt, Chapman, Estillore, Grünewald, Küpper, Laugks, Marlovits, Meents, Oberthür, Samanta, Seuring
Funding: Excellence-Cluster 'Center for Ultrafast Imaging (CUI)’
"High-throughput antibody screening against SARS-CoV2 virus-host interaction utilizing small angle X-ray scattering, electron cryo-microscopy and fast sample delivery"
In this project we will develop methods for the study of the conformational landscape of the Spike protein, and combine cryoEM with SAXS data.
Collaborators: Chapman, Seuring, Svergun, Jeffries, Ayyer, Löw, Nordfeldt
Funding: DESY Strategy Funding
"Cd/Te lambda-Detector (X-Spectrum, DESY)"
Develop microED with on-campus built detectors.
Collaborators: Seuring, Grünewald, Pearson, Chapman, Bücker
Funding: Excellence-Cluster 'Center for Ultrafast Imaging (CUI)’
Open Positions
Research Associate UHH
The cryo-EM facility is hiring a Research Associate for the project “Cluster of Excellence 'CUI: Advanced Imaging of Matter‘ - Freezing Dynamic Structures of Macromolecules in Time" The research associate is responsible for the research and development on time-resolved electron cryo-microscopy using freezing of liquid jets with a focus on application to biological systems ... More Information
Publications
Any use of the resources at the Multi-User CryoEM facility at CSSB requires acknowledgement in publications as follows: “Part of this work was performed at the Multi-User CryoEM Facility at the Centre for Structural Systems Biology, Hamburg, supported by the Universität Hamburg and DFG grant numbers (INST 152/772-1|152/774-1|152/775-1|152/776-1|152/777-1 FUGG). Any assistance provided by staff should be acknowledged. Facility staff members who have acquired and/or interpreted data on your behalf should be invited to be co-authors on the publication, as is usual practice.
2021
Albers S, Beckert B, Matthies M, Mandava C, Schuster R, Seuring C, Riedner M, Sanyal S, Torda A, Wilson D, Ignatova Z (2021) Repurposing tRNAs for nonsense suppression. Nat Commun 12, 3850, https://doi.org/10.1038/s41467-021-24076-x
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 https://doi.org/10.1038/s41586-021-03517-z
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(1):201-217. doi: 10.1002/pro.3986.
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
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
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
2020
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
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 6ARTN eabc172610.1126/sciadv.abc1726
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
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
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
Dominik Vogel SRT, Emmanuelle R. J. Quemin, Kristina Meier,, Tomas Kouba NG, Carola Busch, Sophia Reindl, Stephan Günther,, Stephen Cusack KGn, Maria Rosenthal (2020) Structural and functional characterization of the Severe fever with thrombocytopenia syndrome virus L protein. BioArxiv https://doi.org/10.1101/2020.03.02.973065
Bunduc CM, Fahrenkamp D, Wald J, Ummels R, Bitter W, Houben ENG, Marlovits TC (2020) Structure and dynamics of the ESX-5 type VII secretion system of Mycobacterium tuberculosis. bioRxiv: 2020.2012.2002.408906 doi: 10.1101/2020.12.02.408906
Beckham KSH, Ritter C, Chojnowski G, Mullapudi E, Rettel M, Savitski MM, Mortensen SA, Kosinski J, Wilmanns M. (2020) Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex bioRxiv 2020.11.17.387225; doi: https://doi.org/10.1101/2020.11.17.387225.
2019
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
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
