In the frame of the iNEXT-Discovery project, the access providing partners EMBL and ALBA organise a 2-day Zoom-workshop about cryo-electron tomography and cryo soft X-ray tomography.
The aim of the workshop is to provide the basics of the techniques, their limitations and opportunities. Well-known international experts will give lectures on basic principles and on applications to a variety of biological questions.
Are you a PhD student or a Postdoc or you simply wish to learn more about cryo-electron tomography and cryo soft X-ray tomography, then please register here to attend the workshop!
Limited number of places available, and registration is on a first come, first served basis. Registrations are now closed.
If after the registration you realise you cannot attend the event, kindly cancel the registration and give someone else the chance to attend the workshop.
Day 1 – April 8, 2021
Day 2 – April 9, 2021
The iNEXT-Discovery consortium brings together structural biology facilities for X-rays, NMR, cryo-EM and macromolecular biophysics, and aims to make these facilities accessible to new user communities, to develop methods through joined research efforts, and to offer better integration between scientific fields and within the field of structural biology through scientific meetings, practical courses, and training workshops.
A major objective of iNEXT-Discovery is providing access to users from the European Union, and to some extent outside Europe, to all participating facilities, but also to make available to all users expert structural biology support, to help non-experts answer exciting scientific questions.
iNEXT-Discovery has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 871037. For additional information on the project, please visit iNEXT-Discovery website.
Day 1 – April 8, 2021 | ||
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Time (CEST) | Talk | Speaker |
09:00 | Welcome and general introduction to the workshop | Julia Mahamid (EMBL) |
Session 1: Concepts | Chair: Julia Mahamid (EMBL) | |
09:15 – 10:15 | Cryo X-ray imaging workflows: getting the best of your sample | Eva Pereiro (Alba) |
10:15 – 11:15 | On a cellular scavenge hunt: subcellular targeting and cryo-ET | Philipp Erdmann (Max Planck Institute of Biochemistry) |
11:15 – 12:15 | Basic concepts in tomography for cryo-SXT and cryo-EM | Michael Elbaum (Weizmann Institute of Science) |
12:15 – 12:45 | Meet the speakers | |
Session 2: Putting molecules and structures in their place! Focusing on techniques | Chair: Jiří Nováček (CEITEC) | |
14:10 – 14:40 | Facilitating in-situ structural analysis using contextual subtomogram averaging | Beata Turonova (EMBL) |
14:40 – 15:20 | Correlative 3D imaging of cells using structured illumination microscopy and soft X-ray tomography | Nina Vyas (Diamond Light Source) |
15:20 – 16:00 | Multiscale models of bacteria cell-cell interactions | Martin Pilhofer (Swiss Federal Institute of Technology) |
16:00 – 16:40 | Single-molecule active control microscopy at cryogenic temperatures for correlative microscopies | Peter Dahlberg (Stanford University) |
16:40 – 17:25 | Meet the speakers |
Day 2 – April 9, 2021 | ||
---|---|---|
Time (CEST) | Talk | Speaker |
09:00 | Welcome and introduction | Maria Harkiolaki (Diamond Light Source) |
Session 3: Putting molecules and structures in their place! Correlative approaches | Chair: Maria Harkiolaki (Diamond Light Source) | |
09:10 – 09:50 | Characterizing mineral-bearing vesicles in sea urchin embryos using cryo soft X-ray microscopy and cryo EM-EDS | Keren Kahil (Weizmann Institute of Science) |
09:50 – 10:30 | Integrative structural cell biology of membrane modulations in the course of virus-host interactions | Kay Grünewald (Centre for Structural System Biology) |
10:30 – 11:10 | A correlative multi-scale imaging approach to visualize SARS-CoV-2 infection in cells | Peijun Zhang (University of Oxford) |
11:10 – 11:50 | 3D cryo X-ray imaging to determine the intracellular location of a potent cytotoxic Iridium compound | Javier Conesa (National Centre for Biotechnolgy, Spanish National Research Council) |
11:50 – 12:20 | Meet the speakers | |
14:00 | Introduction to session 4 | Simone Mattei (EMBL) |
Session 4: Instrumentation and Methods | Chair: Simone Mattei (EMBL) | |
14:10 – 14:50 | Advanced instrumentation technology and methods for cryo-electron tomography | Juergen Plitzko (Max Planck Institute of Biochemistry) |
14:50 – 15:30 | Challenges: Measurement of optical and data resolution in SXT. Optimisation of cryo EM subtomogram averaging analysis in Relion | Joaquín Otón (MRC Laboratory of Molecular Biology) |
15:30 – 16:10 | Using the Warp–RELION–M pipeline to obtain high-resolution structures in situ | Dimitry Tegunov (Max Planck Institute for Biophysical Chemistry) |
16:10 – 16:20 | Closing remarks | Eva Pereiro (ALBA) Julia Mahamid (EMBL) |
16:20 – 16:50 | Meet the speakers |
The third party online platform, Zoom, will be used to host our online workshop. We are not liable for problems that occur as a result of use of such platforms. By registering for this online workshop, you agree to accept the providers’ terms and conditions.
Participation can be affected by local issues, such as internet access, but we are not responsible for resolving these issues. We do not provide technical support and each attendee is responsible for setting up their equipment. We suggest you download the Zoom App as it is more reliable and stable than the web browser.
Any links to external resources or materials sent by speakers or other attendees via the platform are opened at the attendees’ own risk. We are not liable for any issues occurring from opening these links.
This event will be recorded and freely available online.
Questions during and after the talks can be asked via the chat. The chair moderates the questions and shares them with the speaker. If time runs out or you think of a question later, you can use the Meet the speakers sessions to ask your questions.
Please do:
Please don’t:
Javier Conesa
National Centre for Biotechnology, Spanish National Research Council
3D cryo X-ray imaging to determine the intracellular location of a potent cytotoxic Iridium compound
The iridium half-sandwich complex we investigated is highly cytotoxic: 15-250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X-ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X-ray tomography (cryo-SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X-ray fluorescence tomography (cryo-XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo-3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape.
Peter Dahlberg
Stanford University
Single-molecule active control microscopy at cryogenic temperatures for correlative microscopies
Single-molecule based super-resolution fluorescence imaging encompasses a broad range of different methods that rely on two key aspects. First, the ability to control emission from fluorescent labels that densely decorate a structure so that at any given time emission from a diffraction limited volume is dominated by an individual emitter. Second, the ability to precisely localize individual emitters based on the point-spread-function generated by their fluorescence emission. The ability to control the emission state of these molecules is so crucial to these methods that we often refer to them by the umbrella term “Single-Molecule Active Control Microscopy” or SMACM for short. Here I will give an overview of SMACM and highlight the challenges present when adapting SMACM to cryogenic temperatures for correlative based microscopies. Most notably, achieving the requisite control under cryogenic conditions in a manner compatible with SXT and CET presents numerous challenges and opportunities. I will discuss our recent work achieving a level of control under cryogenic conditions which has enabled us to precisely and accurately annotate cryogenic tomographic reconstructions with single-molecule fluorescence localizations.
Michael Elbaum
Weizmann Institute of Science
Basic concepts in tomography for cryo-SXT and cryo-EM
Tomography is the art of the 3 R’s: record, reconstruct, and render. Classical tomography is the reconstruction of a 3D volume from 2D projections, based on the Radon transform and its inverse. Another sort of 3D reconstruction is based on serial sections or serial surface images, for example in the focused ion beam – scanning electron microscope (FIB-SEM). This could be called a Cartesian tomography. With an emphasis on cryo-imaging, the talk will present the basic principles underlying each of the three elements: what do we record, what are the options for reconstruction, and how do we render or represent the volume data for interpretation?
Philipp Erdmann
Max Planck Institute of Biochemistry
On A Cellular Scavenge Hunt: Subcellular Targeting and cryo-ET
Life takes place on length scales of considerably different magnitudes. Accordingly, integrative modeling of cellular processes requires techniques that can cover the same length scales. In situ cryo-electron tomography (cryo-ET) in particular can be used to image intact cells in their native state and at high resolution, thus enabling both quantitative analysis of cellular components and modeling of dynamic cellular processes at the molecular level. However, some structures might be rare or confined to specific sites within a cell. They must therefore be targeted during sample preparation to avoid being missed. 3D-correlative cryo-fluorescence microscopy (FLM), automated focused ion beam (FIB) milling, and advanced image processing are just a few building blocks for studying such rare biological events. Here we demonstrate the steps and techniques necessary to prepare biological samples for in situ cryo-ET, with targets ranging from general cellular structures to specific sites on the way to a ‘biopsy at the nanoscale’.
Kay Grünewald
Centre for Structural System Biology
Integrative structural cell biology of membrane modulations in the course of virus-host interactions.
A mechanistic understanding of the complexity of structural cell biology in virus-host interactions requires a combination of tools and approaches. We apply electron cryo tomography (cryoET) in combination with complementary techniques to provide a comprehensive spatio-temporal picture of the functional interaction between viral protein complexes and cellular structures in the course of the infection. Understanding the entirety of a virus’ ‘life cycle’ requires an understanding of its transient structures at the molecular level in their native cellular environment. Viruses serve moreover as dedicated tools to mine the molecular detail of cellular tomograms and to highlight uncharted mechanisms. Members from the herpesviruses, a family of enveloped large DNA viruses, constitute our main model systems. We are particularly interested on steps involving membrane remodelling and report here on the processes of herpesvirus entry (overcoming the plasma membrane) and herpesvirus nuclear egress (i.e. overcoming the double membrane nuclear barrier). Along our biological questions, we constantly expand technologies and workflows and explore new combinations of approaches. The presentation will highlight some of these, including correlative microscopies, super-resolution fluorescence cryo microscopy, X-ray microscopy/tomography and signpost DNA origami nanostructure tags (SPOTs) as novel tools for identifying molecules of interest in complex tomograms.
Keren Kahil
Weizmann Institute of Science
Characterizing Mineral-Bearing Vesicles in Sea Urchin Embryos Using Cryo-Soft X-Ray Microscopy and Cryo-EM-EDS
Keren Kahil, Steve Weiner, Lia Addadi
Sea urchin larvae have endoskeletons comprised of two calcitic spicules. The spicule-forming cells (PMCs) must uptake, process and deposit large amounts of calcium ions in order to facilitate the rapid growth of the spicules. PMCs uptake much of the needed calcium by endocytosis of sea water into a complex network of vacuoles and vesicles. The calcium ions accumulate, and subsequently deposit as amorphous CaCO3 (ACC) that is transferred to the spicule, where it crystallizes. Our study focuses on calcium concentration and storage during the process of mineral formation. We use cryo-soft X-ray tomography correlated with spectro-microscopy (Ca L¬2,3 XANES) to locate and characterize both the phases and the concentrations of Ca-rich bodies in sea urchin larval cells. PMCs contain hundreds of Ca-rich particles, 200-500 nm in diameter. Using the pre-peak /main peak (L2′/ L2) intensity ratio of the XANES spectra, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. We also determined the concentration of Ca in each of the particles by the integrated area in the main Ca absorption peak. We observed ~ 700 Ca-rich particles with order parameters ranging in a continuum from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. These data shed light on intracellular transport and concentration pathways of calcium ions, and in particular on ACC formation in vivo. The proposed cellular process of gradual calcium concentration and ordering may represent a widespread pathway in mineralizing organisms, eventually leading to a deeper understanding of mineral formation.
Joaquín Otón
MRC Laboratory of Molecular Biology
Challenges: Measurement of optical and data resolution in SXT. Optimisation of cryoEM subtomogram averaging analysis in Relion
I present the image formation process in a soft Xray microscope and how the condenser and objective lenses determine the optical parameters of degree of coherence, resolution and depth of field. I also revisit the method to estimate such optical parameters based on a Siemens star test pattern and other methods to estimate the resolution in reconstructed tomography data. Finally, I present my current developments in the optimisation of the computational process for cryoEM subtomogram averaging technique.
Eva Pereiro
Alba
Cryo X-ray imaging workflows: getting the best of your sample
3D X-ray imaging techniques based on absorption, phase contrast and X-ray fluorescence cover a wide range of sample dimensions (from tissue to cells) and resolution, spanning from few tens of nanometers to microns. They can provide structural and chemical information, and can be combined in complex approaches including visible light fluorescence microscopy. Among these techniques, cryo soft X-ray tomography (SXT) can reveal the structural organization of whole hydrated cells with chemical sensitivity. We will give an X-ray imaging overview first before focusing on cryo-SXT and the sample preparation steps required.
Martin Pilhofer
Swiss Federal Institute of Technology
Multiscale models of bacterial cell-cell interactions
We investigate macromolecular machines mediating bacterial cell-cell interactions, by integrating information from the molecular to the cellular and intercellular scale. Our key technique is cryo-electron tomography, applied in an interdisciplinary approach. Our main focus is to understand the function, mechanism and evolution of contractile injection systems.
Juergen Plitzko
Max Planck Institute of Biochemistry
Advanced instrumentation technology and methods for cryo-electron tomography
Recent technological developments for cryo-transmission electron microscopy include cold field emitters, phase plates (hole-free and laser), energy filters and electron detectors. For sample preparation, new methods such as preparation robots, cryo-FIB instruments (using gallium ions as well as plasma ions), off-line and in-line cryo-light microscopy approaches, and furthermore software for automating various workflows and streamlining the analysis of the acquired data have been made available. The basics and the more standard approaches will be covered during this workshop. The focus of my presentation is on the emerging technologies, their working principles and in particular their future-oriented applications for cryo-electron tomography. The emphasis is on the WHAT, WHY, and HOW: what will be their benefits and suitability, why do we need them or not, and how much effort does it take to adopt these tools and techniques. I look forward to meeting you in the virtual space to share both my ideas and my insights into the various and latest developments for cryo-electron tomography.
Dimitry Tegunov
Max Planck Institute for Biophysical Chemistry
Using the Warp–RELION–M pipeline to obtain high-resolution structures in situ
While single-particle analysis of in vitro samples made major advances over the past decade to achieve atomic resolution, the analysis pipeline for tomographic in situ data has lagged behind. With the recent development of Warp and M, we were able to close the gap in processing, obtaining residue-level resolution for ribosomes images inside cells for the first time. In my talk, I will highlight our recent results and the underlying ideas, and go through all steps from tilt series acquisition to obtaining a high-resolution reconstruction.
Beata Turonova
European Molecular Biology Laboratory
Facilitating in-situ structural analysis using contextual subtomogram averaging
Cryo Electron Tomography is essential (you can add “has become essential tool” here) in structural analysis of large macromolecular complexes in their close to native environment. In combination with Subtomogram Averaging one can routinely obtain structures of complexes with subnanometer resolution. In the recent past a lot of effort has been channeled into pushing the technique and thereby the achievable resolution even further. Most of this effort focuses on improving the methodology on subtomogram averaging level, neglecting the valuable contextual information provided within the tomograms. Here, I would like to focus on techniques that use this information to facilitate the subtomogram averaging and further structural analysis on the whole tomogram level.
Nina Vyas
Diamond Light Source
Correlative 3D Imaging of Cells using Structured Illumination Microscopy and Soft X-ray Tomography
Biological imaging has developed rapidly in recent years across scale and resolution ranges but to capture the intricate interplay of structures and processes within living cells different imaging methods need to work together. At the biological cryo-imaging beamline B24 at the UK synchrotron, Diamond Light Source, two high resolution 3D imaging systems have been developed side by side to enable the in depth examination of biological systems at near physiological states. The two imaging methods are: soft X-ray tomography (SXT) which uses the natural absorption contrast of hydrated biological material, such as cells and tissues, to deliver 3D data to 25nm in resolution and fluorescence structured illumination microscopy (SIM) which highlights localisation of tagged molecules, organelles and other cellular structures within the cellular maps captured through X-ray imaging. The fully commissioned workflow starts with samples first imaged in SIM to generate 3D fluorescence information at high resolution and identify areas of interest before they are loaded to the transmission X-ray microscope for SXT data collection on these same areas of interest. This way data recorded at different microscopes can be directly correlated enabling the unambiguous interpretation of data. The beamline workflow will be presented with examples of recent data collected along with highlights and pitfalls of the correlative scheme employed.
Peijun Zhang
University of Oxford
A Correlative Multi-Scale Imaging Approach to Visualize SARS-CoV-2 Infection in Cells
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events – e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus – in the context of whole-cell images. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
Eva Pereiro
ALBA, Barcelona
Julia Mahamid
EMBL, Heidelberg
Simone Mattei
EMBL, Heidelberg
Maria Harkiolaki
Diamond Light Source, Didcot
Bruno Klaholz
Centre for Integrative Biology, Strasbourg
Julia Mahamid
EMBL, Heidelberg
Simone Mattei
EMBL, Heidelberg
Jiri Novacek
CEITEC, Brno
Eva Pereiro
ALBA, Barcelona