This curated selection of life sciences research publications highlight recent work accomplished with cryo-EM single particle analysis and cryo-tomography. Read these noteworthy publications to learn more about how our customers are using Thermo Scientific electron microscopes to advance their research.

Joppe, M. et al. The resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase.

IUCrJ 7, 220–227 (2020). DOI:10.1107/S2052252519017366

This research paper demonstrates that a protocol consisting of protein purification, protein quality monitoring, data collection, and image analysis can be completed within a day. The last two steps took only 8 hours and yielded a 3.1 Å resolution map of the 2.6 MDa yeast fatty acid synthase. While this protocol might not directly apply to other protein complexes, the authors note that their optimization and monitoring of the critical purification step might serve as a blueprint for others.

Thermo Fisher Scientific Instruments used: Tecnai Spirit, Vitrobot Mark IV, Krios, Falcon 3EC, EPU

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Joppe, M. et al. The resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase.
Structural analysis of yeast FAS.

Rougé, L. et al. Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

Science 367, 1224 (2020). DOI:10.1126/science.aaz9356

Rituximab was the first monoclonal antibody approved for the treatment of cancer in 1997. Rituximab targets the B cell membrane protein CD20 for which neither structure nor function was known. The authors determined the structure of CD20 in complex with rituximab and show that they form circular super-assemblies, which might explain how the monoclonal antibody initiates complement-dependent cytotoxicity in B cells. The function of CD20 itself, however, remains an open question since the structure does not support the suggestion of being a cation channel.

Thermo Fisher Scientific Instruments used: Talos F200C, Ceta, Vitrobot Mark IV, Krios

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O’Reilly, F. J. et al. In-cell architecture of an actively transcribing-translating expressome.  

bioRixiv (2020). DOI: 10.1126/science.aba3526

The "expressome" supercomplex consists of RNA polymerase, the ribosome, and two transcription elongation factors. Combining in-cell crosslinking mass spectrometry, in situ cryo-electron tomography and integrated modeling, the authors reveal the structural heterogeneity behind the functional coupling of transcription and translation in bacteria. Using a novel sub-tomogram averaging workflow, O'Reilly et al. were also able to determine the 5.6 Å resolution structure of the 70S ribsome in its native environment.

Thermo Fisher Scientific Instruments used: Fusion Lumos Tribrid, Krios

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Hervas, R. et al. Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila.

 

Science 367, 1230 (2020). DOI: 10.1126/science.aba3526

Amyloids, aggregates formed by misfolded proteins, are usually associated with the disruption of normal cell function in several diseases. But amyloids don’t have to be pathogenic. In neurons of Drosophila melanogaster for example, the protein Orb2 can form aggregates that are implicated in memory formation, consolidation and recall. To investigate the Orb2 aggregates, the authors isolated the biochemically active amyloid from millions of fly heads and determined the filament structure using single particle cryo-EM. They also showed that Orb2 can reduce or enhance translation depending on its form, suggesting that “amyloids could be a stable yet regulatable substrate of memory”.

Thermo Fisher Scientific Instruments used: LTQ, Tecnai 12, Talos L120C, Ceta, Vitrobot Mark IV; Krios, EPU

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Hurdiss, D. L. et al. Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans.

bioRxiv (2020). DOI:10.1101/2020.03.25.998963

HKU1 is one of the coronavirus species circulating in humans and belongs to the genus Betacoronavirus like SARS-CoV, MERS-CoV, and SARS-CoV-2. An infection with HKU1 usually leads to symptoms of the common cold but may also cause severe respiratory disease in some patients. HKU1, as a member of the subgenus Embecovirus, has an additional surface protein called haemagglutinin esterase (HE) which makes members of this subgenus structurally distinct from other coronaviruses. HKU1 HE is heavily glycosylated and has resisted crystallization and structure determination using X-ray crystallography. Despite its small size (~80 kDa), the authors successfully determined its structure using single particle cryo-EM and mass spectrometry. Comparing these data to OC43, another Embecovirus circulating in humans, offers insights into evolution of the HE protein. Furthermore, the data reveal the conserved HE esterase active site which could be a viable target for the development of antivirals to HKU1, OC43 and novel species of this subgenus should they arise.

Thermo Fisher Scientific Instruments used: Vitrobot Mark IV, Krios G4, Falcon 4, EPU, Orbitrap Fusion Tribrid

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Liang, Y.-L. et al. Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins.

ACS Pharmacol. Transl. Sci. (2020). DOI:10.1021/acsptsci.9b00080

The peptide hormones Adrenomedullin (AM1) and Adrenomedullin 2 (AM2) are relevant for normal physiology, development, and disease. Their receptors are heterodimers formed by the calcitonin receptor-like receptor (CLR) combined with either receptor activity-modifying protein (RAMP) 2 or RAMP3 to form AM1 and AM2 receptors. The authors report structures of these receptors in complex with Gs and AM1 or AM2 with resolutions ranging from 2.3 to 3.0 Å. Using 3D multivariate analysis, they were able to resolve some of the molecular motions in the receptor structures that govern phenotype and efficacy of these GPCR receptors.

Thermo Fisher Scientific Instruments used: Vitrobot Mark IV; Krios

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Liang, Y.-L. et al. Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins.
This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
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