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In this episode of the Epigenetics Podcast, we talked with Yali Dou from Keck School of Medicine of USC about her work on MLL Proteins in Mixed-Lineage Leukemia. To start off this Interview Yali describes her early work on MLL1 and its function in transcription, particularly its involvement in histone modification. She explains her successful purification of the MLL complex and the discovery of MOF as one of the proteins involved. Next, the interview focuses on her work in reconstituting the MLL core complex and the insights gained from this process. She shares her experience of reconstituting the MLL complex and discusses her focus on the crosstalk of H3K4 and H3K79 methylation, regulated by H2BK34 ubiquitination. The podcast then delves into the therapeutic potential of MLL1, leading to the discovery of a small molecule inhibitor. Finally, we talk about the importance of the protein WDR5 in the assembly of MLL complexes and how targeting the WDR5-ML interaction can inhibit MLL activity. References Dou, Y., Milne, T., Ruthenburg, A. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat Struct Mol Biol 13, 713–719 (2006). https://doi.org/10.1038/nsmb1128 Wu, L., Zee, B. M., Wang, Y., Garcia, B. A., & Dou, Y. (2011). The RING Finger Protein MSL2 in the MOF Complex Is an E3 Ubiquitin Ligase for H2B K34 and Is Involved in Crosstalk with H3 K4 and K79 Methylation. Molecular Cell, 43(1), 132–144. https://doi.org/10.1016/j.molcel.2011.05.015 Cao, F., Townsend, E. C., Karatas, H., Xu, J., Li, L., Lee, S., Liu, L., Chen, Y., Ouillette, P., Zhu, J., Hess, J. L., Atadja, P., Lei, M., Qin, Z. S., Malek, S., Wang, S., & Dou, Y. (2014). Targeting MLL1 H3K4 Methyltransferase Activity in Mixed-Lineage Leukemia. Molecular Cell, 53(2), 247–261. https://doi.org/10.1016/j.molcel.2013.12.001 Park, S.H., Ayoub, A., Lee, YT. et al. Cryo-EM structure of the human MLL1 core complex bound to the nucleosome. Nat Commun 10, 5540 (2019). https://doi.org/10.1038/s41467-019-13550-2 Related Episodes Dosage Compensation in Drosophila (Asifa Akhtar) Targeting COMPASS to Cure Childhood Leukemia (Ali Shilatifard) Contact Epigenetics Podcast on X Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Epigenetics Podcast on Bluesky Epigenetics Podcast on Threads Active Motif on X Active Motif on LinkedIn Email: podcast@activemotif.com
Everyone thinks microbes are very small, and most of them are. But how to see them? The microscope opened a whole new world to the observer, starting with the Dutch microbiologist Antonie van Leeuvenhoek. But photographs and peering through lenses have limitations. Mark introduces his friend and colleague, Ariane Briegel of the Institute of Biology at Leiden University to Matters Microbial. She discusses how her own work can allow us to see microbes at extremely fine detail using a technique called cryo-electron microscopy (cryEM). She will also discuss her path in science. Host: Mark O. Martin Guest: Ariane Briegel Subscribe: Apple Podcasts, Spotify Become a patron of Matters Microbial! Links for this episode The Martian meteorite from which my specimen was taken is described here. Custom enamel pins by Hartiful can be found here. The website of the great microbiologist and science artist Lizah van der Aart is here. Here is a video discussing the role played by van Leeuvenhoek in microbial science that is SO worth your time. Dr. Briegel's lab website is very interesting. An explainer about cryoEM can be found here A really fine talk by Dr. Briegel about her work from ASM Microbe a few years ago. Intro music is by Reber Clark Send your questions and comments to mattersmicrobial@gmail.com
#12 — Rhys Grinter is Lab Head in the Department of Microbiology at Monash University. In this episode of Cryo-Talk, Rhys joins Eva Amsen to talk about how he uses cryoEM to look at bacterial proteins, including an enzyme that converts air to electricity. They also talk about travel, career breaks, and cooking.Watch or listen to all episodes of the Cryo-Talk podcast here: https://cryo-talk.bitesizebio.com
#11 — Peter Shen is an Assistant Professor of Biochemistry at the University of Utah. In this episode of Cryo-Talk, Peter joins Eva Amsen to talk about the CryoEM 101 course he co-developed and how this led to merit badges for the National Centers for CryoEM. They also talk about boardgames, basketball, and making music. Watch or listen to all episodes of the Cryo-Talk podcast here: https://cryo-talk.bitesizebio.com
#61 — Joachim Frank is a Professor of Biological Sciences at Columbia University and winner of the 2017 Nobel Prize in Chemistry for his involvement in the development of CryoEM. In this episode of The Microscopists, Joachim joins Peter O'Toole to discuss how his early interactions with an electron microscope shaped his career and how he considered moving into environmental research. They also chat about Joachim's passion for writing literary fiction.Watch or listen to all episodes of The Microscopists: http://themicroscopists.bitesizebio.com/
#10 — Gökhan Tolun is an Associate Professor in the School of Chemistry and Molecular Bioscience at the University of Wollongong in Australia, and Research Group Leader at the Molecular Horizons Research Institute. In this episode of Cryo-Talk, Gökhan joins Eva Amsen to talk about his research, funding challenges in different countries, and how Molecular Horizons' new facility was built with microscopy in mind. They also talk about photography, archery, and the two-body problem in science. Watch or listen to all episodes of the Cryo-Talk podcast here: cryo-talk.bitesizebio.com
#9 — Bret Freudenthal is Associate Professor in the Department of Biochemistry at the University of Kansas Medical Center. In this episode of Cryo-Talk, Bret joins Eva Amsen to talk about the importance of making CryoEM technologies accessible via shared facilities and the new facility opening in Kansas. They also talk about skiing, barbecuing, and ‘90s rap music. Watch or listen to all episodes of the Cryo-Talk podcast here: cryo-talk.bitesizebio.com
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536725v1?rss=1 Authors: Tarutani, A., Lövestam, S., Zhang, X., Kotecha, A., Robinson, A. C., Mann, D. M. A., Saito, Y., Murayama, S., Tomita, T., Goedert, M., Scheres, S., Hasegawa, M. Abstract: The formation of amyloid filaments through templated seeding is believed to underlie the propagation of pathology in most human neurodegenerative diseases. A widely used model system to study this process is to seed amyloid filament formation in cultured cells using human brain extracts. Here, we report the electron cryo-microscopy (cryo-EM) structures of tau filaments from undifferentiated seeded SH-SY5Y cells, that transiently expressed N-terminally HA-tagged 1N3R or 1N4R human tau, using brain extracts from individuals with AD or CBD. Although the resulting filament structures differed from those of the brain seeds, some degree of structural templating was observed. Studying templated seeding in cultured cells, and determining the structures of the resulting filaments, can thus provide insights into the cellular aspects underlying neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
#8 — Ariane Briegel is Professor of Ultrastructural Biology at Leiden University and co-director of the Netherlands Centre for Electron Nanoscopy. In this episode of Cryo-Talk, Ariane joins Eva Amsen to share how she uses cryo-electron tomography to study how microbes interact with their environment. They also talk about Ariane's initial interest in marine biology and horseback riding. Watch or listen to all episodes of the Cryo-Talk podcast here: cryo-talk.bitesizebio.com
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534981v1?rss=1 Authors: Zielinski, M., Reyes, F. S. P., Gremer, L., Schemmert, S., Frieg, B., Willuweit, A., Donner, L., Elvers, M., Nilsson, L. N. G., Syvänen, S., Sehlin, D., Ingelsson, M., Willbold, D., Schröder, G. F. Abstract: The development of novel drugs for Alzheimer's disease has proven difficult, with a high failure rate in clinical trials. Typically, transgenic mice displaying amyloid-{beta} peptide brain pathology are used to develop therapeutic options and to test their efficacy in preclinical studies. However, the properties of A{beta} in such mice have not been systematically compared to A{beta} from the patient brains. Here, we determined the structures of nine ex vivo A{beta} fibrils from six different mouse models by cryo-EM. We found novel A{beta} fibril structures in the APP/PS1, ARTE10, and tg-SwDI models, whereas the human familial type II fibril fold was found in the ARTE10, tg-APPSwe, and APP23 models. The tg-APPArcSwe mice showed an A{beta} fibril whose structure resembles the human sporadic type I fibril. These structural elucidations are key to the selection of adequate mouse models for the development of novel plaque-targeting therapeutics and PET imaging tracers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
#7 — Rob Kirchdoerfer is Assistant Professor in the Department of Biochemistry and the Institute for Molecular Virology at the University of Wisconsin-Madison. In this episode of CryoTalk, Rob joins Eva Amsen to talk about using cryoEM to study virus interactions and how he ended up working on cutting-edge research. He also talks about possible future cryoEM applications, why he has been interested in science since he was a kid, and winter in Wisconsin. Tune in to hear more!Watch or listen to all episodes of the Cryo-Talk podcast here: cryo-talk.bitesizebio.com
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.03.522595v1?rss=1 Authors: Prevost, M. S., Barilone, N., Dejean de la Batie, G., Pons, S., Ayme, G., England, P., Gielen, M., Bontems, F., Pehau-Arnaudet, G., Maskos, U., Lafaye, P., Corringer, P.-J. Abstract: The human 7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest to design ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of 7 ligands, we recently generated two nanobodies named E3 and C4 acting as positive and silent allosteric modulators respectively. Here, we solved the cryo-EM structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an active or desensitized conformation in the absence of orthosteric agonist, and mutational analysis shows a key contribution of a N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation which opens new avenues for drug design. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.15.520545v1?rss=1 Authors: Shi, Y., Ghetti, B., Goedert, M., Scheres, S. Abstract: Positron emission tomography (PET) imaging allows monitoring the progression of amyloid aggregation in the living brain. [18F]-Flortaucipir is the only approved PET tracer compound for the visualisation of tau aggregation. Here, we describe cryo-EM experiments on tau filaments in the presence and absence of flortaucipir. We used tau filaments isolated from the brain of an individual with Alzheimer's disease (AD), and from the brain of an individual with primary age-related tauopathy (PART) with a co-pathology of chronic traumatic encephalopathy (CTE). Unexpectedly, we were unable to visualise additional cryo-EM density for flortaucipir for AD paired helical or straight filaments (PHFs or SFs), but we did observe density for flortaucipir binding to CTE Type I filaments from the case with PART. In the latter, flortaucipir binds in a 1:1 molecular stoichiometry with tau, adjacent to lysine 353 and aspartate 358. By adopting a tilted geometry with respect to the helical axis, the 4.7 A distance between neighbouring tau monomers is reconciled with the 3.5 A distance consistent with pi-pi-stacking between neighbouring molecules of flortaucipir. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.10.519870v1?rss=1 Authors: Fung, H. K., Hayashi, Y., Salo, V. T., Babenko, A., Zagoriy, I., Brunner, A., Ellenberg, J., Mueller, C. W., Cuylen-Haering, S., Mahamid, J. Abstract: Cryo-electron tomography is a powerful label-free tool for visualizing biomolecules in their native cellular context at molecular resolution. However, the precise localisation of biomolecules of interest in the tomographic volumes is challenging. Here, we present a tagging strategy for intracellular protein localisation based on genetically encoded multimeric particles (GEMs). We show the applicability of drug-controlled GEM labelling of endogenous proteins in cryo-electron tomography and cryo-correlative fluorescence imaging in human cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.25.517942v1?rss=1 Authors: Wu, D., Dai, Y., Gao, N. Abstract: Bacterial HflX is a conserved ribosome-binding GTPase involved in splitting ribosomal complexes accumulated under stress condition. However, the atomic details of its ribosomal interaction remain to be elucidated. In this work, we present a high-resolution structure of the E. coli 50S subunit bound with HflX. The structure reveals highly specific contacts between HflX and the ribosomal RNA, and in particular, an insertion loop of the N-terminal domain of HflX is situated in the peptidyl transferase center (PTC) and makes direct interactions with PTC residues. Interestingly, this loop displays steric clash with a few PTC-targeting antibiotics on the 50S subunit, such as chloramphenicol. Deletion of hflX results in hypersensitivity to chloramphenicol treatment, and a loop residue G154 of HflX is important for the observed chloramphenicol resistance. Overall, our results suggest that HflX could be a general stress response factor that functions in both stalled ribosome splitting and PTC antibiotic displacing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.24.517842v1?rss=1 Authors: Ralhan, I., Chang, J., Moulton, M. J., Goodman, L. D., Lee, N. Y., Plummer, G., Pasolli, H. A., Matthies, D., Bellen, H. J., Ioannou, M. S. Abstract: During oxidative stress neurons release lipids that are internalized by glia. Defects in this coordinated process play an important role in several neurodegenerative diseases. Yet, the mechanisms of lipid release and its consequences on neuronal health are unclear. Here, we demonstrate that lipid-protein particle release by autolysosome exocytosis protects neurons from ferroptosis, a form of cell death driven by lipid peroxidation. We show that during oxidative stress, peroxidated lipids and iron are released from neurons by autolysosomal exocytosis which requires the exocytic machinery; VAMP7 and syntaxin 4. We observe membrane-bound lipid-protein particles by TEM and demonstrate that these particles are released from neurons using cryoEM. Failure to release these lipid-protein particles causes lipid hydroperoxide and iron accumulation and sensitizes neurons to ferroptosis. Our results reveal how neurons protect themselves from peroxidated lipids. Given the number of brain pathologies that involve ferroptosis, defects in this pathway likely play a key role in the pathophysiology of neurodegenerative disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
#6 — Mimi Ho is Assistant Professor of Microbiology and Immunology at Columbia University. In this episode of Cryo-Talk, Mimi joins Eva Amsen to talk about her career journey from industry to academia, her support network, and how Mighty the dog has been helping in the lab. She also shares what it has been like to co-host The Plunge podcast. Tune in now to hear more!
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.08.515609v1?rss=1 Authors: Leistner, C., Wilkinson, M., Burgess, A., Goodbody, S., Xu, Y., Deuchars, S., Radford, S. E., Ranson, N. A., Frank, R. A. W. Abstract: Amyloid plaques composed of extracellular focal deposition of A{beta} fibrils are a hallmark of Alzheimer's disease (AD). Cryo-EM structures of A{beta} fibrils purified from human AD brain tissue post mortem have recently been determined. However, the molecular architecture of amyloid plaques in the context of fresh, unfixed mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the native, in situ molecular architecture of A{beta} fibrils in the brain of a mouse model containing the Arctic familial AD mutation (AppNL-G-F) and an atomic model of Arctic A{beta} fibril purified from the brains of these animals. We show that in-tissue A{beta} fibrils are arranged in a lattice or in parallel bundles within a plaque, and are interdigitated by subcellular compartments, exosomes, extracellular droplets and extracellular multilamellar bodies. At the atomic level, the Arctic A{beta} fibril differs significantly from earlier structures of A{beta} amyloid extracted from AppNL-F mice models and human AD brain tissue, showing a striking effect of the Arctic mutation (E22G) on fibril structure. Cryo-electron tomography of ex vivo purified and in-tissue amyloid revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises {beta}-amyloid plaque pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.07.515410v1?rss=1 Authors: Yang, Y., Zhang, W., Murzin, A. G., Schweighauser, M., Huang, M., Lovestam, S., Peak-Chew, S. Y., Saito, T., Saido, T. C., McDonald, J., Lavenir, I., Ghetti, B., Graff, C., Kumar, A., Nordberg, A., Goedert, M., Scheres, S. H. Abstract: The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-beta] (Abeta)], causes dominantly inherited Alzheimer's disease. Here we report the high-resolution cryo-EM structures of A beta filaments from the frontal cortex of a previously described case (A beta PParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12-V40 (human Arctic fold A) and E11-G37 (human Arctic fold B). They have a substructure (residues F20-G37) in common with the folds of type I and type II Abeta42. When compared to the structures of wild-type Abeta42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Abeta molecules and promote filament formation. A minority of Abeta42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Abeta filaments with the Arctic mutation from mouse knock-in line App NL-G-F. Most filaments are made of two identical mutant protofilaments that extend from D1-G37 (murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The murine Arctic fold differs from the human Arctic folds, but shares some substructure. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
⚡ Cryo-EM is a powerful tool that helps look at cancer molecules differently. Penn State University uses the cryo-EM technique to understand and outsmart cancer. Professor Kelly explains, "Our lab uses a very high-tech imaging approach. It's called cryo-electron microscopy or cryo-EM, which pioneers in our field actually won the Nobel Prize for just a few years ago. And what we'd like to do is dive deep into cancer cells, understand what molecules look like using these instruments, take pictures and snapshots of them — what you would do with your iPhone but in portrait mode — so we can really focus very deeply on the nuances of these molecules. Then we use these molecules to try and better understand what goes wrong in cancer, how these molecules are to cancer, and what we might do to better inform treatments based on differences in molecules from cancer cells versus normal cells."⚡ Cryo-electron microscopy allows us to image things at the level of atoms. So what makes cryo-EM technology so useful in cancer research? Professor Kelly says, "What cryo-EM does is it allows us to see all the molecules that constitute cells, their different placements within cells, as well as their over architecture down at the level of atoms. So going even deeper beyond just the level of cells, we can get down and understand the level of which proteins are with DNA, how these proteins don't interact with DNA properly to protect cells from diseases, or how things might work against us when cells become cancerous and how molecules go awry and don't perform their job properly."⚡ What makes Penn State unique in cryo-EM? Professor Kelly explains what makes her lab's cryo-EM one of a kind. She says, "Cryo-electron microscopes that are installed and operational at Penn State are uniquely built to service the life science community as well as the material science community. And some of these instruments have different analytical tools and cameras integrated in them that you wouldn't find in any other cryo-EM instrument. We're looking to screen and look at proteins differently."
#5 — Mike Cianfrocco is Research Assistant Professor at the University of Michigan Life Sciences Institute and Assistant Professor in the Department of Biological Chemistry at the University of Michigan Medical School. In this episode of Cryo-Talk, Mike joins Eva Amsen to talk about the tools he is developing for cryoEM users, such as COSMIC2. He also chats about his love of gardening and fermented foods. Tune in now to hear more!
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.18.512754v1?rss=1 Authors: Stern, A. M., Yang, Y., Meunier, A. L., Liu, W., Cai, Y., Ericsson, M., Liu, L., Goedert, M., Scheres, S. H., Selkoe, D. J. Abstract: Soluble aggregates of amyloid-{beta} (A{beta}), often called oligomers, are believed to be principal drivers of neurotoxicity, spreading of pathology, and symptoms in Alzheimers disease (AD), but little is known about their structures in human brain. A{beta} oligomers have been defined as aggregates found in supernatants following ultracentrifugation of aqueous extracts. We now report the unexpected presence of abundant A{beta} fibrils in high-speed supernatants from AD brains that were extracted by soaking in aqueous buffer. The fibrils did not appear to form during extract preparation, and their numbers by EM correlated with ELISA quantification of aggregated A{beta}42. Cryo-EM structures of A{beta} fibrils from aqueous extracts were identical to those from sarkosyl-insoluble AD brain homogenates. The fibrils in aqueous extracts were immunolabeled by lecanemab, an A{beta} aggregate-directed antibody reported to improve cognitive outcomes in AD. We conclude that A{beta} fibrils are abundant in aqueous extracts from AD brains and have the same structures as those from amyloid plaques. These findings have implications for understanding the nature of A{beta} oligomers and for designing oligomer-preferring therapeutic antibodies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
#52 — Wah Chiu is Wallenberg Bienenstock Professor, and Professor of Bioengineering, and of Microbiology and Immunology at Stanford University. In this episode of the Microscopists, Wah chats with Peter O' Toole about his pioneering cryoEM work and his research goals of understanding the structural biology of organelles. They also discuss careers in academia versus industry, the role of AI and alpha fold in structural biology, and speculate on the future of microscopy. On a lighter note, they chat about the importance of keeping fit, seeing family, and taking a break on holiday—and touch on being productive on plane journeys and city hopping across Europe. Watch all The Microscopists episodes here: http://bit.ly/the-microscopists-yt #TheMicroscopists #Imaging #CryoEM
#52 — Wah Chiu is Wallenberg Bienenstock Professor, and Professor of Bioengineering, and Microbiology and Immunology at Stanford University. In this episode of the Microscopists, Wah chats with Peter O' Toole about his pioneering cryoEM work and his research goals of understanding the structural biology of organelles.They also discuss careers in academia versus industry, the role of AI and alpha fold in structural biology, and speculate on the future of microscopy. On a lighter note, they chat about the importance of keeping fit, seeing family, and taking a break on holiday—and touch on being productive on plane journeys and city hopping across Europe.Watch or Listen to all episodes of The Microscopists here: https://themicroscopists.bitesizebio.com/
#4 — Liz Kellogg is Assistant Professor in the Department of Molecular Biology and Genetics at Cornell University. In this episode of Cryo-Talk, Liz joins Eva Amsen to share how she uses cryoEM to learn more about CRISPR-associated transposons. We also hear about the challenges of keeping a new lab going during the early days of COVID and find out what her favorite music is. Tune in to hear more!
#26 — Cryo-EM is a revolutionary imaging method that lets us see complex biostructures at higher and higher resolutions. But do you understand the mind-blowing science behind this technique? And what is cryo-electron microscopy, anyway? Why is the cryogenic aspect important, and how did it seemingly go from nothing to the big time? In the latest episode of Mentors At Your Benchside, we answer all of these questions and more! Check out the corresponding online article to access loads of follow-up resources to deepen your understanding of this topic.[1] Also, check out our related articles covering crucial sample preparation considerations for cryo-EM and its history from obscure to Nobel Prize winner. [2,3] Resources: 1. What Is Cryo-Electron Microscopy? Available at: https://bitesizebio.com/62871/what-is-cryo-electron-microscopy/ 2. Cryo-EM Sample Prep: 5 Crucial Considerations. Available at: https://bitesizebio.com/62619/cryo-em-sample-prep/ 3. A Short History of Cryo-Electron Microscopy: Available at: https://bitesizebio.com/62839/history-of-cryo-electron-microscopy/
This week, Natalie and Tiffany chat with Rose Marie Haynes, microscopist at the Pacific Northwest Center for Cryo-EM and Chair of Professional Development here at WISPDX! We talked about the gradual change we see in inclusivity in STEM, how physics is made cool, and how our relationships with science should grow and evolve as we do. Rose Marie Haynes uses she/her pronouns and works as a microscopist, where she works at the intersection of physics, chemistry and biology to use advanced instrumentation to determine biological structures. In 2019 she graduated with an MS in Applied Physics with a specialization in optical materials and devices. When she's not playing with microscopes or working on programming and events for WIS, she enjoys competitive dancing and knitting matching clothes for her dog and cat. You can email us at podcast@womeninsciencepdx.org and follow us @women_in_science_pdx on Instagram, Twitter, and Facebook.
In dieser Episode besprechen Daniel Roderer und Bernd Rupp die unterschiedlichen Rollen des Mikrobioms. Dabei werden auch potenziell schädliche Mechanismen gezeigt und wie Daniel dieser "dunklen Seite des Mikrobioms" mit Cryo-EM auf der Spur ist.
#3 — Yiorgo Skiniotis of Stanford University has been using cryoEM to study transmembrane receptors. In this episode of Cryo-Talk, Yiorgo joins Eva Amsen to chat about the potential of cryoEM to gather more information about signaling pathways. We also hear more about his love of cinema and classic literature, why he'd be a fisherman if he had to pick another job, and why it's so important to have various research interests. Tune in to hear more!
#2 — Eva Nogales of UC Berkeley and Lawrence Berkeley National Laboratory uses cryoEM to study cellular processes related to cytoskeletal self-assembly and gene expression. In this episode of Cryo-Talk, Eva joins our host Eva Amsen to discuss the use of CryoEM to study complex cell biology systems and more. She chats about her current work while on sabbatical at CNIO in Spain, what music she likes, and her love of books. We also hear why she thinks it's so important to work with people that you get along with. Tune in to hear more!
Cryo-EM is groundbreaking in the field of structural biology, allowing researchers to get a better look at complex proteins. This is valuable for studying any kind of proteins that are related to any kind of human disease.Now, UC has the technology to do it.
In this episode we dive deep into the world of structural biology. We discuss the role of cryogenic electron microscopy in the development of precision medicines with the founder and CEO of Gandeeva Therapeutics, Dr. Sriram Subramaniam. Cryo-EM opened a new way to study drug-target interactions and is changing the way we develop new therapies.Tune into our interview with Sriram to learn more about: ◦ The founding story of Gandeeva Therapeutics ◦ The evolution of Cryo-EM technology ◦ Application of Cryo-EM in drug discovery ◦ Advantages of Cryo-EM over crystallography im mapping protein structures ◦ Making sense of experimental data with AI and machine learning ◦ The future of personalized medicine and role of structural biology in it ◦ Sriram's advice for budding life science entrepreneurs
Alfredo De Biasio, Assistant Professor, Bioscience, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, SAUDI ARABIA speaks on "Human DNA replication under the microscope: Visualizing the lagging strand replisome at high-resolution using cryo-EM".
#43 — Elizabeth Villa, a Howard Hughes Medical Institute investigator at UC San Diego talks to Peter O'Toole about the benefits of collaborative projects, the advantages and disadvantages of new microscopy techniques, and establishing fun lab traditions. We'll chat about her early career as a Fulbright Scholar, her movement into biology to work with microscopy rock stars in the US and Europe, and understanding the social side of proteins using Cryo-EM. Watch or Listen to all episodes of The Microscopists here: http://bit.ly/the-microscopists-yt #TheMicroscopists #microscopy #imageanalysis
#43 — Elizabeth Villa, a Howard Hughes Medical Institute investigator at UC San Diego talks to Peter O'Toole about the benefits of collaborative projects, the advantages and disadvantages of new microscopy techniques, and establishing fun lab traditions. We'll chat about her early career as a Fulbright Scholar, her movement into biology to work with microscopy rock stars in the US and Europe, and understanding the social side of proteins using Cryo-EM.Watch or Listen to all episodes of The Microscopists here: https://themicroscopists.bitesizebio.com/
#1 — Joachim Frank of Columbia University has spent his career working on EM and cryoEM. In this episode of Cryo-Talk, Joachim joins Eva Amsen to discuss his research and his 2017 Nobel Prize in Chemistry. We'll hear how he has used peripheral vision to find unexpected opportunities, why he loves fiction writing, and how he balances New York City life with his time in the Berkshires. We also learn about his Master's project studying the back-scattering of electrons on liquid gold, his first post-doc at the Jet Propulsion Laboratory, and the conversation turns to butterflies on multiple occasions. Tune in to hear more!
特定のRNAの発現に反応する細胞内センサーについての原著論文を紹介しました。Show notes A split ribozyme that links detection of a native RNA to orthogonal protein outputs. BioRxiv 2022 … 今回紹介するプレプリントの論文。 57. All papers are created equal - Researchat.fm … “科学論文の探し方、読み方とその楽しみ、そして理想の論文について三人で熱っぽく話しました” Ribozyme (Wikipedia) Tetrahymena Ribozyme … テトラヒメナ由来のリボザイムの反応についての解説。 Thomas Cech (Wikipedia) … “RNAが触媒的機能を持ち細胞内での反応に関与していることを初めて発見し、これをリボザイムと命名し…1989年にノーベル化学賞を受賞した” Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 A resolution. Nature 2021 … テトラヒメナの立体構造解析を行った論文。 Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature 1994 Raman2RNA: Live-cell label-free prediction of single-cell RNA expression profiles by Raman microscopy. BioRxiv 2021 Editorial notes この分野はおそらくこの先も面白い技術が次々に出てきそうです (soh) まぁええんじゃないですか (tadasu)
NOTA: este episodio se grabó el 2 de marzo de 2020, justo unos días antes del confinamiento por la pandemia Covid19. Por su relevancia en aquellos días de gran incertidumbre, inmediatamente se editó tan solo un fragmento del programa, la entrevista a Ignacio López-Goñi, y se publicó como episodio BS11. El resto de la grabación debería haberse publicado como episodio BS12 pero la situación de alerta y el confinamiento que fue decretado pocos días después, hizo que se quedara sin editar y, por tanto, sin publicar. De hecho, no volvimos a grabar hasta finales del 2020 (episodios BS13-14, grabados por videoconferencia). Este episodio BS12, recuperado ahora, se grabó, como era -y, afortunadamente, es ya también- habitual en el estudio de grabación de la Facultad de Ciencias de la Comunicación (Universidad de Málaga). Tras las efemérides del día y la bienvenida, comenzamos (min. 02:55) comentando en la tertulia la situación de alerta mundial por coronavirus y la difusión de la información relacionada con este asunto ( recuérdese que estábamos en marzo de 2020). A continuación (min. 16:36), Pepe nos presenta un artículo que acababa de publicar el grupo de Marcos Malumbres (@m_malumbres, Centro Nacional de investigaciones Oncológicas, Madrid, lab webpage: https://malumbreslab.org) en el que demuestran que la combinación de quimioterapia (taxol) e inhibidores de CDK4/6 (en ese orden) es efectiva contra uno de los tumores más agresivos: el adenocarcinoma de páncreas (la referencia es: Salvador-Barbero et al. (2020). CDK4/6 Inhibitors Impair Recovery from Cytotoxic Chemotherapy in Pancreatic Adenocarcinoma. Cancer Cell 37, 340-353.e6. https://doi.org/10.1016/j.ccell.2020.01.007). Para conocer mejor los detalles del trabajo, entrevistamos al propio Dr. Malumbres (min. 28:20). Tras la entrevista, damos paso a la sección de Bionoticias (min. 37:32). En esta ocasión, nuestros reporteros más dicharacheros, Belén Delgado e Íker Puerto escogieron dos noticias destacadas: la celebración del Día Mundial de las Enfermedades Raras (29 de febrero) y el anuncio del descubrimiento de nuevos antibióticos mediante estrategias computacionales de Deep Learning (la referencia es: Stokes et al. (2020). A Deep Learning Approach to Antibiotic Discovery. Cell 180, 688-702.e13. https://doi.org/10.1016/j.cell.2020.01.021). A partir del minuto 63:20, Francis nos presenta su selección particular: uno de los artículos clave en el que se describe la estructura de la proteína espicular del coronavirus SarsCov2, obtenida por CryoEM en el laboratorio del Dr. Jason McLellan (la referencia es: Wrapp et al. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260–1263. https://doi.org/10.1126/science.abb2507). En la sección “El libro de la semana” entrevistamos a la investigadora Andrea Martos (@andreamesteban, University of Cambridge, blog: https://andreamartosesteban.medium.com), autora del libro “Volver a hacer fiestas”, un original ensayo sobre las herramientas de edición CRISPR-Cas, con el que ganó el 1er Certamen de Ensayo de Divulgación Científica de la Universidad Autónoma de Madrid (UAM). Como siempre, esperamos que os guste este episodio (especialmente, creemos que disfrutaréis las dos entrevistas) y que os parezca interesante. Un saludo, y gracias por seguir ahí. ------------------ Resumen (minuto y contenido): 0:00 Efemérides y bienvenida 02:55 Tertulia. Comentamos la alerta mundial (marzo 2020) por coronavirus. 16:36 Artículo Pepe: Quimioterapia combinada con inhibidores CDK4/6 en cáncer de páncreas (https://doi.org/10.1016/j.ccell.2020.01.007) 28:20 Entrevista a Marcos Malumbres (CNIO, Madrid) 37:32 Bionoticias, con Íker y Belén: Día Mundial de las Enfermedades Raras (29 de febrero) y el descubrimiento de antibióticos mediante Deep Learning 63:20 Artículo Francis: estructura de la proteína espicular del coronavirus SarsCov2, (https://doi.org/10.1126/science.abb2507) 74:20 El libro de la semana: entrevista a Andrea Martos, autora de “Volver a hacer fiestas”, un original ensayo sobre las herramientas de edición CRISPR-Cas. 107:30 Despedida. 👍
Welcome to a new BioPOD series: Scotland's Biotech Stories. In this installment, BioPodder Liz Gaberdiel interviews Dr. Marcus Wilson on Cryogenic electron microscopy (CryoEM), a technique that has undergone some serious upgrades since its initial development in the 1960s. Introduction by Neelakshi Varma & Editing by Sam Haynes Media by Hanna Peach and Chris Donohoe
Linoleic acid is an essential free fatty acid in the human body and its metabolic pathway is central to immune regulation and inflammation – which are also key symptoms in COVID-19. Using cryo-electron microscopy, Christine Toelzer’s research identified linoleic acid bound to a hydrophobic pocket of the SARS-CoV-2 glycoprotein. Christine shares her thoughts on how these findings will contribute to the fight against COVID-19 and how her lab work has been altered by the pandemic. Christine also discusses the future of other young scientists coming up in the protein science space. Christine Toelzer is currently a Research Associate at the University of Bristol. After a M.Sc. in biology and an additional M.Sc. in physics she continued with PhD work in biochemistry at the University of Cologne. Her research has always focused on structure function relationships, starting with structure determination of biotechnologically important proteins by x-ray crystallography, magnetic structure determination of inorganic compounds by neutron diffraction and recently using electron cryo-microscopy to obtain the structure of large protein complexes involved in transcription and diseases. In the last year (2020) she started coronavirus related work to contribute to the global effort aimed at better understanding the virus and uncover its potential weaknesses.About the Young Scientist Keynote Award:This recognition honors a young scientist from the international protein science community who has contributed to scientific advancement and innovation in this field. Nominations were solicited from across academic and industry research groups in the fall of 2020, and the finalists were determined through the votes and input of our 15-person advisory panel.
Listen to Dr. Eva Nogales describe how cryo-electron microscopy addresses the challenge of visualizing macromolecular structures.
Byl to vypečený rok, a proto jej shrnujeme ve vypečené sestavě s kolegy popularizátory – řeč přijde jak na covid a věci kolem něj ( a konspirace :(), tak i na doslova závody o vesmír. A možná dojde i na další velké objevy! Prosvištíme si: Cryo EM jako mikroskopie budoucnosti Supravodivost za pokojové teploty Covid-19 pandemii a vakcíny co nás z ní vysekají Konspirace k 5G a k tomu dřívějšímu prů*eru Převratné úspěchy SpaceX Útok na Mars a prastaré planetky
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387068v1?rss=1 Authors: Malhotra, S., Joseph, A. P., Thiyagalingam, J., Topf, M. Abstract: Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for the map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we have assessed protein-protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed PI-score, a density-independent machine learning-based metric, trained using protein-protein interfaces features in high-resolution crystal structures. Using PI-score, we were able to identify errors at interfaces in the PDB-deposited cryo-EM structures (including SARS-CoV-2 complexes) and in the models submitted for cryo-EM targets in CASP13 and the EM model challenge. Some of the identified errors, especially at medium-to-low resolution structures, were not captured by density-based assessment scores. Our method can therefore provide a powerful complementary assessment tool for the increasing number of complexes solved by cryo-EM. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.376822v1?rss=1 Authors: Walter, J. D., Hutter, C. A. J., Garaeva, A. A., Scherer, M., Zimmermann, I., Wyss, M., Rheinberger, J., Ruedin, Y., Earp, J. C., Egloff, P., Sorgenfrei, M., Hürlimann, L., Gonda, I., Meier, G., Remm, S., Thavarasah, S., Zimmer, G., Slotboom, D. J., Paulino, C., Plattet, P., Seeger, M. A. Abstract: The COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. Cryo-EM analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. In the symmetric complex each of the three RBDs were bound by both sybodies, and adopted the up conformation. The asymmetric conformation, with three Sb#15 and two Sb#68 bound, contained one down RBD, one up-out RBD and one up RBD. Bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. Our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent SARS-CoV-2 inhibitors for potential therapeutic applications. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.08.372763v1?rss=1 Authors: Sun, M., Azumaya, C. M., Tse, E., Frost, A., Southworth, D., Verba, K. A., Cheng, Y., Agard, D. A. Abstract: Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed nonCDS mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 [A] with a 1.346 [A] physical pixel). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 [A] physical pixel size) we achieved a resolution of 1.65 [A] within 9 hours of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 [A] resolution with 5 minutes of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.05.370247v1?rss=1 Authors: Wang, R. Y.- R., Noddings, C. M., Kirschke, E., Myasnikov, A., Johnson, J. L., Agard, D. A. Abstract: Maintaining a healthy proteome is fundamental for organism survival. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90's many "client" proteins. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop "loading" complex, conversion to an active GR:Hsp90:p23 "maturation" complex, and subsequent GR release. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s--one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.05.369835v1?rss=1 Authors: Deng, Z., Zhao, Y., Feng, J., Zhang, J., Zhao, H., Rau, M. J., Fitzpatrick, J., Hu, H., Yuan, P. Abstract: TMEM206 has been recently identified as an evolutionarily conserved chloride channel that underlies ubiquitously expressed, proton-activated, outwardly rectifying anion currents. Here we report the cryo-electron microscopy structure of pufferfish TMEM206, which forms a trimeric channel, with each subunit comprising two transmembrane segments, the outer and inner helices, and a large extracellular domain. An ample vestibule in the extracellular region is accessible laterally from the three side portals. The central pore contains multiple constrictions preventing ion conduction. A conserved lysine residue near the cytoplasmic end of the inner helix forms the presumed chloride ion selectivity filter. Unprecedentedly, the core structure and assembly closely resemble those of the epithelial sodium channel/degenerin family of sodium channels that are unrelated in amino acid sequence and conduct cations instead of anions. Together with electrophysiology, this work provides insights into ion conduction and gating for a new class of chloride channels that is architecturally distinct from previously characterized chloride channel families. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.04.368837v1?rss=1 Authors: Liu, Y., Sun, M., Elnatan, D., Larson, A. G., Agard, D. A. Abstract: Hsp90 is a ubiquitous molecular chaperone that mediates the folding and maturation of hundreds of ''client'' proteins. Although Hsp90s generally function as homodimers, recent discoveries suggested that the mitochondrion specific Hsp90 (TRAP1) also forms functionally relevant tetramers. The structural mechanism of tetramer formation remains elusive. Here we used a combination of solution, biochemical and cryo-electron microscopy (cryo-EM) approaches to confirm that, independent of nucleotide state, a subpopulation of TRAP1 exists as tetramers. Unexpectedly, cryo-EM reveals multiple tetramer conformations having TRAP1 dimers arranged in orthogonal, parallel, or antiparallel configurations. The cryo-EM structure of one of the orthogonal tetrameric states was determined at 3.5 [A] resolution. Each of the two TRAP1 dimers is in a symmetric AMP{middle dot}PNP-bound closed state with the tetramer being stabilized through three distinct dimer-dimer interaction sites. In unique ways, each of the three TRAP1 domains contributes to tetramer formation. In addition to tetramerization via direct dimer-dimer contacts, our structure suggests that additional stabilization could come from domain swapping between the dimers. These results expand our understanding of TRAP1 biology beyond the conventional view of a functional dimer and provide a platform to further explore the function and regulation of tetrameric TRAP1 in mitochondria. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.03.364851v1?rss=1 Authors: Lizarrondo, J., Klebl, D. P., Niebling, S., Abella, M., Schroer, M. A., Mertens, H. D. T., Veith, K., Svergun, D. I., Skruzny, M., Sobott, F., Muench, S., Garcia-Alai, M. M. Abstract: During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites. Copy rights belong to original authors. Visit the link for more info
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