The evolution of 200 kV cryo EM
Cryo-electron microscopy (cryo-EM) has come a long way since the first viral structures were published by the Dubochet group in the early 1980s. Year over year, there is an almost exponential increase in the number of cryo-EM structures submitted to the Protein Data Bank (PDB), many at resolutions <3 Å. Notably, these structures are often for membrane proteins and other systems that have been resistant to traditional crystallographic analysis. In this blog post, learn how technology, including the new 200 kV Thermo Scientific Glacios 2 Cryo-TEM, has evolved, and see some of the early and impressive results already accomplished by Thermo Fisher Scientific’s newest model announced in October 2022.
Realizing potential with the new Glacios 2 Cryo-TEM
The vast majority of <3 Å structures submitted to the PDB were determined with 300 kV cryo-transmission electron microscopes (cryo-TEMs) that were specifically designed for high-resolution data collection at cryogenic temperatures. In fact, it is safe to say that 300 kV has been treated as the de facto accelerating voltage for high quality structural analysis. When they were introduced, 200 kV instruments such as the Thermo Scientific Talos Arctica and Glacios Cryo-TEMs were, therefore, largely seen as screening tools that could be used to optimize samples without spending time on the higher-resolution 300 kV instruments.
However, advanced components and software are not reserved exclusively for 300 kV microscopes; they have allowed 200 kV cryo-TEMs to experience their own “resolution revolution,” and research groups around the world are beginning to produce highly valuable results directly at 200 kV.
#EMBOMPs now with pic👇🏽
Couldn't be more proud & thrilled to see @chancie_t show our results (@MembraneEnzymo1) on the ECF transporter and the use of 200kV #cryoEM – not a screening machine! Very deserved he got to talk right after the amazing pioneers Brian Kobilka & @gonenlab pic.twitter.com/Vzfqy7HIgv
— Cristina Paulino (@CPaulino_) December 1, 2021
Prof. Cristina Paulino at the University of Groningen highlighting her group’s development of an optimized cryo-EM data-acquisition workflow by sample-thickness determination. Learn more >>
A revolution in productivity and cryo-EM resolution
The new Glacios 2 Cryo-TEM showcases many of the advances that have made high-quality analysis possible on 200 kV instruments. Hardware optimization has focused on improved specificity and sensitivity in electron detection. For instance, the Thermo Scientific Falcon 4i Direct Electron Detector enables high detective quantum efficiency (DQE) across the full spectral range, capturing both small/flexible proteins as well as larger structures using fewer images. When coupled with the novel Thermo Scientific Selectris or Selectris X Energy Filters, signal is limited to the zero-loss, elastically scattered electrons, producing high-contrast TEM data for higher throughput and higher resolution structures.
With the introduction of Thermo Scientific Smart EPU Software, both automated screening and data acquisition for single particle cryo-EM analysis are now available through an approachable graphical user interface. Featuring unique AI algorithms, Smart EPU Software allows for thousands of images to be collected reproducibly and with minimal user input, and integrates aberration free image shift (AFIS), , producing more accurate, useable data with each scan.
What follows are just a few examples of how 200 kV cryo-TEM is being utilized by researchers internationally to produce novel, actionable results.
200keV can be used for high resolution cryoEM: aldolase (150kda) at ~2.6 Å on a Talos Arctica. https://t.co/DgzVrzzyMw pic.twitter.com/UOTQuQOp9r
— Lander Lab (@LanderLab) May 25, 2017
Prof. Gabe Lander’s group at the Scripps Research Institute celebrates the publication of their ~2.6 Å resolution structure of aldolase (150kda) in 2017 and later published a ∼1.75 Å resolution structure of apoferritin in 2020, both determined with 200 kV cryo-TEM. Learn more >>
Cryo-EM of human fucosidase
The enzyme fucosidase plays a vital role in certain bacterial infections as well as fucosidosis, a neurodegenerative disorder impacting lysosomal storage. A complete understanding of fucosidase catalysis, along with any potential targeted drug design, has been hampered by the lack of 3D structures for the enzyme. Utilizing a Glacios Cryo-TEM, researchers at the University of York were able to resolve the structure of human lysosomal α-L-fucosidase (FucA1) down to 2.49 Å resolution. Not only that, but they were also able to clearly visualize FucA1 in complex with an inhibitor (deoxyfuconojirimycin). At this level of detail, the precise architecture of the catalytic center could be determined as well as the location of various disease-related mutations. This serves as a promising step toward targeted treatment of fucosidase-related diseases and disorders.
I've been away from twitter with life stuff but wanted to take a moment to share the first bio paper from the @YSBL_York Glacios. All of the data was collected in-house on our Falcon-IV and it's a beautiful enzyme! I'll never tire of the 2D classes https://t.co/Xop8NGMwVJ pic.twitter.com/63r5ZsdCgr
— Jamie Blaza (@JNB_lab) August 2, 2022
Jamie Blaza, a contributor on the article, shares some of the beautiful 2D classes they obtained for FucA1.
Drug discovery at 200 kV
Structure-based drug design has been a longstanding goal of pharmaceutical research, as it enables the development of targeted treatments based on molecular insights. Efforts have largely been hampered by the speed at which actionable data can be collected and processed. Modern cryo-EM, however, has made significant strides toward structure-enabled therapeutics thanks to high-throughput, high-resolution data.
For example, Prof. Basil Greber of the Institute of Cancer Research (London) has used the Glacios 2 Cryo-TEM to determine the structure of a CDK-activating kinase (CAK). These enzymes are involved in cell regulation and are actively being investigated as potential targets for cancer treatments.
Watch the on-demand webinar: Harnessing the power of cryo-EM for discovery of cancer therapeutics >>
GPCR structure determination
G protein-coupled receptors (GPCRs) compose a large class of membrane proteins that are promising therapeutic targets for a variety of diseases, but they are notoriously difficult to crystallize, hampering crystallographic characterization. Cryo-EM has made significant headway in the analysis of GPCRs in recent years, but this work has been limited to 300 kV instruments. However, researchers at Monash University have been able to successfully determine the structure of a GPCR to 3.2 Å resolution on a 200 kV Glacios Cryo-TEM, a platform whose general ease of use and increased throughput has the potential to significantly broaden structure-based drug design for GPCRs.
200 kV advancements beyond single particle cryo-EM: MicroED of organometallics
Microcrystal electron diffraction (microED) is a burgeoning small molecule characterization technique propelled by the increased availability of high-resolution cryo-electron microscopy equipment. MicroED can analyze much smaller samples (i.e., nanocrystals) compared to X-ray diffraction, and, unlike other electron diffraction techniques, is designed to be compatible with sensitive organic samples such as protein crystals.
We've come a long way since our first EPU-D training session back in April last year… The Glacios has really transformed the way we're able to collect electron diffraction data so it's great to have this instrument in house!
— Emily Thompson (@EmilyA_Thompson) February 4, 2022
A recent study from the University of York showcases how 200 kV cryo-TEM can be used for the MicroED analysis of single crystal organometallics. Specifically, single crystal to single crystal (SC-SC) transformations, performed on grid, can be
used to generate and characterize otherwise highly reactive organometallic species. Here, nanocrystals composed of a norbornadiene complex were hydrogenated by the addition of H2 gas into a σ-alkane (norbornane) complex. The resulting crystallographic structures were resolved down to 0.95 Å resolution. These results not only showcase the quality of MicroED data that can be generated at 200 kV, but more broadly demonstrate the potential of cryo-EM for on-grid single-crystal chemistry.
Cryo-electron tomography on the new Glacios 2 Cryo-TEM
Tomography is a reconstructive imaging technique that combines multiple 2D snapshots collected at different angles into a single 3D dataset. Cryo-electron tomography (cryo-ET), specifically, is used on thinned cell samples to obtain high-resolution 3D information on organelles and proteins within their cellular context. Using the Glacios 2 Cryo-TEM, tomography data can be obtained from whole bacterial cells or lamellae prepared with Thermo Scientific Aquilos or new Arctis Cryo-Focused Ion Beam (Cryo-FIB) instruments. In the Magnetospirillum example shown below, membranous compartments, filaments, and larger proteins can clearly be visualized within the 300 nm thick plunge-frozen bacterial cell.
The future of 200 kV cryo-EM
Cryo-EM software and hardware continue to improve at an astonishing pace, painting a bright future for the technique. Notably, cutting-edge data processing, along with AI-driven modeling of cryo-EM data, is showing incredible potential for taking us from sample to structure faster than ever before.
Another #ModelAngelo success! It managed to build 80% of the very loopy structural model into the 3.3A Glacios 200 keV map. Although may not look impressive at first, it allowed me to align individual AlphaFold predicted domains and fit perfectly. Full model done in 20 minutes https://t.co/lodV9rziAb pic.twitter.com/G7Go1xzHBM
— Martin Pacesa (@MartinPacesa) October 24, 2022
Martin Pacesa of EPFL shows the development of a 3.3 Å reconstruction in under an hour using the novel Model Angelo modeling tool.
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