Podcasts about cryo em

 If you're a scientist, and you apply for federal research funding, you'll ask for a specific dollar amount. Let's say you're asking for a million-dollar grant. Your grant covers the direct costs, things like the salaries of the researchers that you're paying. If you get that grant, your university might get an extra $500,000. That money is called “indirect costs,” but think of it as overhead: that money goes to lab space, to shared equipment, and so on.This is the system we've used to fund American research infrastructure for more than 60 years. But earlier this year, the Trump administration proposed capping these payments at just 15% of direct costs, way lower than current indirect cost rates. There are legal questions about whether the admin can do that. But if it does, it would force universities to fundamentally rethink how they do science.The indirect costs system is pretty opaque from the outside. Is the admin right to try and slash these indirect costs? Where does all that money go? And if we want to change how we fund research overhead, what are the alternatives? How do you design a research system to incentivize the research you actually wanna see in the world?I'm joined today by Pierre Azoulay from MIT Sloan and Dan Gross from Duke's Fuqua School of Business. Together with Bhaven Sampat at Johns Hopkins, they conducted the first comprehensive empirical study of how indirect costs actually work. Earlier this year, I worked with them to write up that study as a more accessible policy brief for IFP. They've assembled data on over 350 research institutions, and they found some striking results. While negotiated rates often exceed 50-60%, universities actually receive much less, due to built-in caps and exclusions.Moreover, the institutions that would be hit hardest by proposed cuts are those whose research most often leads to new drugs and commercial breakthroughs.Thanks to Katerina Barton, Harry Fletcher-Wood, and Inder Lohla for their help with this episode, and to Beez for her help on the charts.Let's say I'm a researcher at a university and I apply for a federal grant. I'm looking at cancer cells in mice. It will cost me $1 million to do that research — to pay grad students, to buy mice and test tubes. I apply for a grant from the National Institutes of Health, or NIH. Where do indirect costs come in?Dan Gross: Research generally incurs two categories of costs, much as business operations do.* Direct or variable costs are typically project-specific; they include salaries and consumable supplies.* Indirect or fixed costs are not as easily assigned to any particular project. [They include] things like lab space, data and computing resources, biosecurity, keeping the lights on and the buildings cooled and heated — even complying with the regulatory requirements the federal government imposes on researchers. They are the overhead costs of doing research.Pierre Azoulay: You will use those grad students, mice, and test tubes, the direct costs. But you're also using the lab space. You may be using a shared facility where the mice are kept and fed. Pieces of large equipment are shared by many other people to conduct experiments. So those are fixed costs from the standpoint of your research project.Dan: Indirect Cost Recovery (ICR) is how the federal government has been paying for the fixed cost of research for the past 60 years. This has been done by paying universities institution-specific fixed percentages on top of the direct cost of the research. That's the indirect cost rate. That rate is negotiated by institutions, typically every two to four years, supported by several hundred pages of documentation around its incurred costs over the recent funding cycle.The idea is to compensate federally funded researchers for the investments, infrastructure, and overhead expenses related to the research they perform for the government. Without that funding, universities would have to pay those costs out of pocket and, frankly, many would not be interested or able to do the science the government is funding them to do.Imagine I'm doing my mouse cancer science at MIT, Pierre's parent institution. Some time in the last four years, MIT had this negotiation with the National Institutes of Health to figure out what the MIT reimbursable rate is. But as a researcher, I don't have to worry about what indirect costs are reimbursable. I'm all mouse research, all day.Dan: These rates are as much of a mystery to the researchers as it is to the public. When I was junior faculty, I applied for an external grant from the National Science Foundation (NSF) — you can look up awards folks have won in the award search portal. It doesn't break down indirect and direct cost shares of each grant. You see the total and say, “Wow, this person got $300,000.” Then you go to write your own grant and realize you can only budget about 60% of what you thought, because the rest goes to overhead. It comes as a bit of a shock the first time you apply for grant funding.What goes into the overhead rates? Most researchers and institutions don't have clear visibility into that. The process is so complicated that it's hard even for those who are experts to keep track of all the pieces.Pierre: As an individual researcher applying for a project, you think about the direct costs of your research projects. You're not thinking about the indirect rate. When the research administration of your institution sends the application, it's going to apply the right rates.So I've got this $1 million experiment I want to run on mouse cancer. If I get the grant, the total is $1.5 million. The university takes that .5 million for the indirect costs: the building, the massive microscope we bought last year, and a tiny bit for the janitor. Then I get my $1 million. Is that right?Dan: Duke University has a 61% indirect cost rate. If I propose a grant to the NSF for $100,000 of direct costs — it might be for data, OpenAI API credits, research staff salaries — I would need to budget an extra $61,000 on top for ICR, bringing the total grant to $161,000.My impression is that most federal support for research happens through project-specific grants. It's not these massive institutional block grants. Is that right?Pierre: By and large, there aren't infrastructure grants in the science funding system. There are other things, such as center grants that fund groups of investigators. Sometimes those can get pretty large — the NIH grant for a major cancer center like Dana-Farber could be tens of millions of dollars per year.Dan: In the past, US science funding agencies did provide more funding for infrastructure and the instrumentation that you need to perform research through block grants. In the 1960s, the NSF and the Department of Defense were kicking up major programs to establish new data collection efforts — observatories, radio astronomy, or the Deep Sea Drilling project the NSF ran, collecting core samples from the ocean floor around the world. The Defense Advanced Research Projects Agency (DARPA) — back then the Advanced Research Projects Agency (ARPA) — was investing in nuclear test detection to monitor adherence to nuclear test ban treaties. Some of these were satellite observation methods for atmospheric testing. Some were seismic measurement methods for underground testing. ARPA supported the installation of a network of seismic monitors around the world. Those monitors are responsible for validating tectonic plate theory. Over the next decade, their readings mapped the tectonic plates of the earth. That large-scale investment in research infrastructure is not as common in the US research policy enterprise today.That's fascinating. I learned last year how modern that validation of tectonic plate theory was. Until well into my grandparents' lifetime, we didn't know if tectonic plates existed.Dan: Santi, when were you born?1997.Dan: So I'm a good decade older than you — I was born in 1985. When we were learning tectonic plate theory in the 1990s, it seemed like something everybody had always known. It turns out that it had only been known for maybe 25 years.So there's this idea of federal funding for science as these massive pieces of infrastructure, like the Hubble Telescope. But although projects like that do happen, the median dollar the Feds spend on science today is for an individual grant, not installing seismic monitors all over the globe.Dan: You applied for a grant to fund a specific project, whose contours you've outlined in advance, and we provided the funding to execute that project.Pierre: You want to do some observations at the observatory in Chile, and you are going to need to buy a plane ticket — not first class, not business class, very much economy.Let's move to current events. In February of this year, the NIH announced it was capping indirect cost reimbursement at 15% on all grants.What's the administration's argument here?Pierre: The argument is there are cases where foundations only charge 15% overhead rate on grants — and universities acquiesce to such low rates — and the federal government is entitled to some sort of “most-favored nation” clause where no one pays less in overhead than they pay. That's the argument in this half-a-page notice. It's not much more elaborate than that.The idea is, the Gates Foundation says, “We will give you a grant to do health research and we're only going to pay 15% indirect costs.” Some universities say, “Thank you. We'll do that.” So clearly the universities don't need the extra indirect cost reimbursement?Pierre: I think so.Dan: Whether you can extrapolate from that to federal research funding is a different question, let alone if federal research was funding less research and including even less overhead. Would foundations make up some of the difference, or even continue funding as much research, if the resources provided by the federal government were lower? Those are open questions. Foundations complement federal funding, as opposed to substitute for it, and may be less interested in funding research if it's less productive.What are some reasons that argument might be misguided?Pierre: First, universities don't always say, “Yes” [to a researcher wishing to accept a grant]. At MIT, getting a grant means getting special authorization from the provost. That special authorization is not always forthcoming. The provost has a special fund, presumably funded out of the endowment, that under certain conditions they will dip into to make up for the missing overhead.So you've got some research that, for whatever reason, the federal government won't fund, and the Gates Foundation is only willing to fund it at this low rate, and the university has budgeted a little bit extra for those grants that it still wants.Pierre: That's my understanding. I know that if you're going to get a grant, you're going to have to sit in many meetings and cajole any number of administrators, and you don't always get your way.Second, it's not an apples-to-apples comparison [between federal and foundation grants] because there are ways to budget an item as a direct cost in a foundation grant that the government would consider an indirect cost. So you might budget some fractional access to a facility…Like the mouse microscope I have to use?Pierre: Yes, or some sort of Cryo-EM machine. You end up getting more overhead through the back door.The more fundamental way in which that approach is misguided is that the government wants its infrastructure — that it has contributed to through [past] indirect costs — to be leveraged by other funders. It's already there, it's been paid for, it's sitting idle, and we can get more bang for our buck if we get those additional funders to piggyback on that investment.Dan: That [other funders] might not be interested in funding otherwise.Why wouldn't they be interested in funding it otherwise? What shouldn't the federal government say, “We're going to pay less. If it's important research, somebody else will pay for it.”Dan: We're talking about an economies-of-scale problem. These are fixed costs. The more they're utilized, the more the costs get spread over individual research projects.For the past several decades, the federal government has funded an order of magnitude more university research than private firms or foundations. If you look at NSF survey data, 55% of university R&D is federally funded; 6% is funded by foundations. That is an order of magnitude difference. The federal government has the scale to support and extract value for whatever its goals are for American science.We haven't even started to get into the administrative costs of research. That is part of the public and political discomfort with indirect-cost recovery. The idea that this is money that's going to fund university bloat.I should lay my cards on the table here for readers. There are a ton of problems with the American scientific enterprise as it currently exists. But when you look at studies from a wide range of folks, it's obvious that R&D in American universities is hugely valuable. Federal R&D dollars more than pay for themselves. I want to leave room for all critiques of the scientific ecosystem, of the universities, of individual research ideas. But at this 30,000-foot level, federal R&D dollars are well spent.Dan: The evidence may suggest that, but that's not where the political and public dialogue around science policy is. Again, I'm going to bring in a long arc here. In the 1950s and 1960s, it was, “We're in a race with the Soviet Union. If we want to win this race, we're going to have to take some risky bets.” And the US did. It was more flexible with its investments in university and industrial science, especially related to defense aims. But over time, with the waning of these political pressures and with new budgetary pressures, the tenor shifted from, “Let's take chances” to “Let's make science and other parts of government more accountable.” The undercurrent of Indirect Cost Recovery policy debates has more of this accountability framing.This comes up in this comparison to foundation rates: “Is the government overpaying?” Clearly universities are willing to accept less from foundations. It comes up in this perception that ICR is funding administrative growth that may not be productive or socially efficient. Accountability seems to be a priority in the current day.Where are we right now [August 2025] on that 15% cap on indirect costs?Dan: Recent changes first kicked off on February 7th, when NIH posted its supplemental guidance, that introduced a policy that the direct cost rates that it paid on its grants would be 15% to institutions of higher education. That policy was then adopted by the NSF, the DOD, and the Department of Energy. All of these have gotten held up in court by litigation from universities. Things are stuck in legal limbo. Congress has presented its point of view that, “At least for now, I'd like to keep things as they are.” But this has been an object of controversy long before the current administration even took office in January. I don't think it's going away.Pierre: If I had to guess, the proposal as it first took shape is not what is going to end up being adopted. But the idea that overhead rates are an object of controversy — are too high, and need to be reformed — is going to stay relevant.Dan: Partly that's because it's a complicated issue. Partly there's not a real benchmark of what an appropriate Indirect Cost Recovery policy should be. Any way you try to fund the cost of research, you're going to run into trade-offs. Those are complicated.ICR does draw criticism. People think it's bloated or lacks transparency. We would agree some of these critiques are well-founded. Yet it's also important to remember that ICR pays for facilities and administration. It doesn't just fund administrative costs, which is what people usually associate it with. The share of ICR that goes to administrative costs is legally capped at 26% of direct costs. That cap has been in place since 1991. Many universities have been at that cap for many years — you can see this in public records. So the idea that indirect costs are going up over time, and that that's because of bloat at US universities, has to be incorrect, because the administrative rate has been capped for three decades.Many of those costs are incurred in service of complying with regulations that govern research, including the cost of administering ICR to begin with. Compiling great proposals every two to four years and a new round of negotiations — all of that takes resources. Those are among the things that indirect cost funding reimburses.Even then, universities appear to under-recover their true indirect costs of federally-sponsored research. We have examples from specific universities which have reported detailed numbers. That under-recovery means less incentive to invest in infrastructure, less capacity for innovation, fewer clinical trials. So there's a case to be made that indirect cost funding is too low.Pierre: The bottom line is we don't know if there is under- or over-recovery of indirect costs. There's an incentive for university administrators to claim there's under-recovery. So I take that with a huge grain of salt.Dan: It's ambiguous what a best policy would look like, but this is all to say that, first, public understanding of this complex issue is sometimes a bit murky. Second, a path forward has to embrace the trade-offs that any particular approach to ICR presents.From reading your paper, I got a much better sense that a ton of the administrative bloat of the modern university is responding to federal regulations on research. The average researcher reports spending almost half of their time on paperwork. Some of that is a consequence of the research or grant process; some is regulatory compliance.The other thing, which I want to hear more on, is that research tools seem to be becoming more expensive and complex. So the microscope I'm using today is an order of magnitude more expensive than the microscope I was using in 1950. And you've got to recoup those costs somehow.Pierre: Everything costs more than it used to. Research is subject to Baumol's cost disease. There are areas where there's been productivity gains — software has had an impact.The stakes are high because, if we get this wrong, we're telling researchers that they should bias the type of research they're going to pursue and training that they're going to undergo, with an eye to what is cheaper. If we reduce the overhead rate, we should expect research that has less fixed cost and more variable costs to gain in favor — and research that is more scale-intensive to lose favor. There's no reason for a benevolent social planner to find that a good development. The government should be neutral with respect to the cost structure of research activities. We don't know in advance what's going to be more productive.Wouldn't a critic respond, “We're going to fund a little bit of indirect costs, but we're not going to subsidize stuff that takes huge amounts of overhead. If universities want to build that fancy new telescope because it's valuable, they'll do it.” Why is that wrong when it comes to science funding?Pierre: There's a grain of truth to it.Dan: With what resources though? Who's incentivized to invest in this infrastructure? There's not a paid market for science. Universities can generate some licensing fees from patents that result from science. But those are meager revenue streams, realistically. There are reasons to believe that commercial firms are under-incentivized to invest in basic scientific research. Prior to 1940, the scientific enterprise was dramatically smaller because there wasn't funding the way that there is today. The exigencies of war drew the federal government into funding research in order to win. Then it was productive enough that folks decided we should keep doing it. History and economic logic tells us that you're not going to see as much science — especially in these fixed-cost heavy endeavors — when those resources aren't provided by the public.Pierre: My one possible answer to the question is, “The endowment is going to pay for it.” MIT has an endowment, but many other universities do not. What does that mean for them? The administration also wants to tax the heck out of the endowment.This is a good opportunity to look at the empirical work you guys did in this great paper. As far as I can tell, this was one of the first real looks at what indirect costs rates look like in real life. What did you guys find?Dan: Two decades ago, Pierre and Bhaven began collecting information on universities' historical indirect cost rates. This is a resource that was quietly sitting on the shelf waiting for its day. That day came this past February. Bhaven and Pierre collected information on negotiated ICR rates for the past 60 years. During this project, we also collected the most recent versions of those agreements from university websites to bring the numbers up to the current day.We pulled together data for around 350 universities and other research institutions. Together, they account for around 85% of all NIH research funding over the last 20 years.We looked at their:* Negotiated indirect cost rates, from institutional indirect cost agreements with the government, and their;* Effective rates [how much they actually get when you look at grant payments], using NIH grant funding data.Negotiated cost rates have gone up. That has led to concerns that the overhead cost of research is going up — these claims that it's funding administrative bloat. But our most important finding is that there's a large gap between the sticker rates — the negotiated ICR rates that are visible to the public, and get floated on Twitter as examples of university exorbitance — and the rates that universities are paid in practice, at least on NIH grants; we think it's likely the case for NSF and other agency grants too.An institution's effective ICR funding rates are much, much lower than their negotiated rates and they haven't changed much for 40 years. If you look at NIH's annual budget, the share of grant funding that goes to indirect costs has been roughly constant at 27-28% for a long time. That implies an effective rate of around 40% over direct costs. Even though many institutions have negotiated rates of 50-70%, they usually receive 30-50%.The difference between those negotiated rates and the effective rates seems to be due to limits and exceptions built into NIH grant rules. Those rules exclude some grants, such as training grants, from full indirect cost funding. They also exclude some direct costs from the figure used to calculate ICR rates. The implication is that institutions receive ICR payments based on a smaller portion of their incurred direct costs than typically assumed. As the negotiated direct cost falls, you see a university being paid a higher indirect cost rate off a smaller — modified — direct cost base, to recover the same amount of overhead.Is it that the federal government is saying for more parts of the grant, “We're not going to reimburse that as an indirect cost.”?Dan: This is where we shift a little bit from assessment to speculation. What's excluded from total direct costs? One thing is researcher salaries above a certain level.What is that level? Can you give me a dollar amount?Dan: It's a $225,700 annual salary. There aren't enough people being paid that on these grants for that to explain the difference, especially when you consider that research salaries are being paid to postdocs and grad students.You're looking around the scientists in your institution and thinking, “That's not where the money is”?Dan: It's not, even if you consider Principal Investigators. If you consider postdocs and grad students, it certainly isn't.Dan: My best hunch is that research projects have become more capital-intensive, and only a certain level of expenditure on equipment can be included in the modified total direct cost base. I don't have smoking gun evidence, it's my intuition.In the paper, there's this fascinating chart where you show the institutions that would get hit hardest by a 15% cap tend to be those that do the most valuable medical research. Explain that on this framework. Is it that doing high-quality medical research is capital-intensive?Pierre: We look at all the private-sector patents that build on NIH research. The more a university stands to lose under the administration policy, the more it has contributed over the past 25 years — in research the private sector found relevant in terms of pharmaceutical patents.This is counterintuitive if your whole model of funding for science is, “Let's cut subsidies for the stuff the private sector doesn't care about — all this big equipment.” When you cut those subsidies, what suffers most is the stuff that the private sector likes.Pierre: To me it makes perfect sense. This is the stuff that the private sector would not be willing to invest in on its own. But that research, having come into being, is now a very valuable input into activities that profit-minded investors find interesting and worth taking a risk on.This is the argument for the government to fund basic research?Pierre: That argument has been made at the macro-level forever, but the bibliometric revolution of the past 15 years allows you to look at this at the nano-level. Recently I've been able to look at the history of Ozempic. The main patent cites zero publicly-funded research, but it cites a bunch of patents, including patents taken up by academics. Those cite the foundational research performed by Joel Habener and his team at Massachusetts General Hospital in the early 1980s that elucidated the role of GLP-1 as a potential target. This grant was first awarded to Habener in 1979, was renewed every four or five years, and finally died in 2008, when he moved on to other things. Those chains are complex, but we can now validate the macro picture at this more granular level.Dan: I do want to add one qualification which also suggests some directions for the future. There are things we still can't see — despite Pierre's zeal. Our projections of the consequence of a 15% rate cap are still pretty coarse. We don't know what research might not take place. We don't know what indirect cost categories are exposed, or how universities would reallocate. All those things are going to be difficult to project without a proper experiment.One thing that I would've loved to have more visibility into is, “What is the structure of indirect costs at universities across the country? What share of paid indirect costs are going to administrative expenses? What direct cost categories are being excluded?” We would need a more transparency into the system to know the answers.Does that information have to be proprietary? It's part of negotiations with the federal government about how much the taxpayer will pay for overhead on these grants. Which piece is so special that it can't be shared?Pierre: You are talking to the wrong people here because we're meta-scientists, so our answer is none of it should be private.Dan: But now you have to ask the university lawyers.What would the case from the universities be? “We can't tell the public what we spend subsidy on”?Pierre: My sense is that there are institutions of academia that strike most lay people as completely bizarre.Hard to explain without context?Pierre: People haven't thought about it. They will find it so bizarre that they will typically jump from the odd aspect to, “That must be corruption.” University administrators are hugely attuned to that. So the natural defensive approach is to shroud it in secrecy. This way we don't see how the sausage is made.Dan: Transparency can be a blessing and a curse. More information supports more considered decision-making. It also opens the door to misrepresentation by critics who have their own agendas. Pierre's right: there are some practices that to the public might look unusual — or might be familiar, but one might say, “How is that useful expense?” Even a simple thing like having an administrator who manages a faculty's calendar might seem excessive. Many people manage their own calendars. At the same time, when you think about how someone's time is best used, given their expertise, and heavy investment in specialized human capital, are emails, calendaring, and note-taking the right things for scientists [to be doing]? Scientists spend a large chunk of their time now administering grants. Does it make sense to outsource that and preserve the scientist's time for more science?When you put forward data that shows some share of federal research funding is going to fund administrative costs, at first glance it might look wasteful, yet it might still be productive. But I would be able to make a more considered judgment on a path forward if I had access to more facts, including what indirect costs look like under the hood.One last question: in a world where you guys have the ear of the Senate, political leadership at the NIH, and maybe the universities, what would you be pushing for on indirect costs?Pierre: I've come to think that this indirect cost rate is a second-best institution: terrible and yet superior to many of the alternatives. My favorite alternative would be one where there would be a flat rate applied to direct costs. That would be the average effective rate currently observed — on the order of 40%.You're swapping out this complicated system to — in the end — reimburse universities the same 40%.Pierre: We know there are fixed costs. Those fixed costs need to be paid. We could have an elaborate bureaucratic apparatus to try to get it exactly right, but it's mission impossible. So why don't we give up on that and set a rate that's unlikely to lead to large errors in under- or over-recovery. I'm not particularly attached to 40%. But the 15% that was contemplated seems absurdly low.Dan: In the work we've done, we do lay out different approaches. The 15% rate wouldn't fully cut out the negotiation process: to receive that, you have to document your overhead costs and demonstrate that they reached that level. In any case, it's simplifying. It forces more cost-sharing and maybe more judicious investments by universities. But it's also so low that it's likely to make a significant amount of high-value, life-improving research economically unattractive.The current system is complicated and burdensome. It might encourage investment in less productive things, particularly because universities can get it paid back through future ICR. At the same time, it provides pretty good incentives to take on expensive, high-value research on behalf of the public.I would land on one of two alternatives. One of those is close to what Pierre said, with fixed rates, but varied by institution types: one for universities, one for medical schools, one for independent research institutions — because we do see some variation in their cost structures. We might set those rates around their historical average effective rates, since those haven't changed for quite a long time. If you set different rates for different categories of institution, the more finely you slice the pie, the closer you end up to the current system. So that's why I said maybe, at a very high level, four categories.The other I could imagine is to shift more of these costs “above the line” — to adapt the system to enable more of these indirect costs to be budgeted as direct costs in grants. This isn't always easy, but presumably some things we currently call indirect costs could be accounted for in a direct cost manner. Foundations do it a bit more than the federal government does, so that could be another path forward.There's no silver bullet. Our goal was to try to bring some understanding to this long-running policy debate over how to fund the indirect cost of research and what appropriate rates should be. It's been a recurring question for several decades and now is in the hot seat again. Hopefully through this work, we've been able to help push that dialogue along. This is a public episode. If you would like to discuss this with other subscribers or get access to bonus episodes, visit www.statecraft.pub

⚡Gatekeepers of Strength: Allopurinol, Calcium & the RyR Revolution”!   Did you know that an old gout drug might hold the key to reversing age-related muscle weakness and heart failure? Using cryo-EM, researchers found that allopurinol and xanthine derivatives directly activate ryanodine receptors—calcium channels essential for muscle contraction.  

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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/2020.11.05.369835v1?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/2020.11.05.370247v1?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