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  • richardmitnick 9:34 am on September 19, 2019 Permalink | Reply
    Tags: , , Cryo-electron microscopy, Mona Wong-San Diego Supercomputer Center, Online gateways, , ,   

    From Science Node: Women in STEM-“Don’t worry about the software” Mona Wong 

    Science Node bloc
    From Science Node

    09 Sep, 2019
    Alisa Alering

    Science gateway developer Mona Wong combines passion for programming with love of science.

    Have you ever set out to accomplish a new task but quickly discovered that it required new equipment? And then you had to learn how to use the equipment before you could move on, and before long you were bogged down in online instructional videos and wondering why you ever thought this was a good idea?

    Mona Wong develops online gateways that help scientists reach their goals without worrying about how to install and run complex software.

    That same thing can happen to scientists, particularly when they want to access new technologies to expedite their work. The hurdle can be especially big for researchers in fields like biology or the humanities, that don’t traditionally provide technological training.

    Enter Mona Wong, a software engineer at the San Diego Supercomputer Center (SDSC).

    SDSC Triton HP supercomputer

    SDSC Gordon-Simons supercomputer

    SDSC Dell Comet supercomputer

    Wong works behind-the-scenes to develop online gateways that help scientists reach their goals—whether that’s a better understanding of how the brain works or more accurate predictions of natural disasters.

    A science gateway is a tool that makes data, resources, or custom scientific workflows easier to use via a point-and-click web interface—no command lines or programming languages to learn.

    “Gateways allow people to not have to worry about the software and how to install and run it, to be able to try things,” says Wong. “I get to use what’s fun and easy for me to help scientists discover something.”

    Solving problems for scientists

    One way that Wong helps expand access to science is through her work with the Extended Developer Support (EDS) group at the Science Gateways Community Institute (SGCI). Funded by the National Science Foundation, SGCI is a clearinghouse of support for developing, operating, and sustaining science gateways.

    In her role with EDS, Wong works on multiple gateways—often serving entirely different domains. When someone is interested in building a gateway, they apply to EDS. If they’re selected to receive support, they’re assigned to a developer who then meets with the principal investigator and comes up with a work plan for the engagement.

    One of her recent projects is the COSMIC2 Gateway which focuses on structural biology, a field currently undergoing something of a revolution. Structural biologists use cryo-electron microscopy (cryo-EM) to examine the structures of proteins and other macromolecular samples at near-atomic resolution.

    Cryo-Electron Microscope. No image credit.

    Cryo-EM is an invaluable tool for understanding human health and disease, but its widespread use is hindered by the incredibly large size of the datasets the equipment generates.

    In Cryo-EM, scientists use the microscope to collect images of a frozen specimen at increasing depths. The images are then reconstructed into a 3D representation of the original structure.

    The final images constitute very large datasets, currently up to 12K by 8K for a single image.

    Because the microscopes are very expensive instruments, scientists must wait to be allocated time, and when they get access, they collect as many images as they can, generating massive amounts of data at one time—up to tens of terabytes.

    Many biologists aren’t familiar with handling such large datasets or have the computing resources to do the calculations. That’s where a gateway comes in.

    Resolution revolution. The blob-like area on the left of this cryo-EM composite image of beta-galactosidase would have been considered state-of-the-art just a few years ago. The detailed structure on the right shows >10x greater resolution thanks to technological improvement and advanced computational methods. Courtesy Veronica Falconieri, Subramaniam Lab, National Cancer Institute.

    “The user uploads their data to the gateway and sets up parameters for their computational task,” says Wong. “Once the task is initiated by the user, the gateway will generate and submit the commands to the software running on the supercomputer to perform the calculations. The user is notified when the job is done, and they come back to the gateway to view the results.”

    One additional challenge with such large datasets, is how to reliably transfer the data from the scientist’s lab to the supercomputer that does the heavy lifting and back again.

    “If you have only one gigabyte of data and the transfer dies halfway through, you can start again,” says Wong. But that’s not a great solution when you’re dealing with multiple terabytes that can take days or weeks to transfer. That’s why COSMIC2and other gateways use the Globus platform for secure data transfer.

    “The great thing about Globus is if there’s a problem in the middle, it can restart from there,” says Wong. “At the end, it has mechanisms in place that check the data to make sure it wasn’t corrupted. In theory, you don’t have to worry about it.”

    Make the code do something

    Toiling behind-the-scenes might seem like thankless work, but Wong sees her role as actively supporting scientific discovery. She has always loved science, but her own unique path became clear when she took her first computer science course.

    “I discovered that it was actually easy for me,” she says. “It fits the way I think, and it’s also very creative, because you take code and you make it help you do something.”

    And the ‘something’ she is doing hasn’t gone unnoticed. Wong received the Young Professionals Award at the SGCI’s Gateways 2018 conference, which recognizes notable achievement in the advancement of science gateways.

    “It’s a great honor, a way to acknowledge the work that you do and the promise you hold for the community,” says Wong. “But I also sense a kind of responsibility.”

    That responsibility isn’t just to scientists. The use of science gateways is expanding as more research areas are discovering what can be accomplished with access to supercomputing power. Their ease of use allows even school children to learn about advanced scientific resources without worrying about how to install and run complex software—which just makes Wong’s work all the more rewarding.

    “I love that gateways are used a lot now in teaching students about science,” says Wong. “I love science, so I think that the more people who love science, the better.”

    See the full article here .

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    Science Node is an international weekly online publication that covers distributed computing and the research it enables.

    “We report on all aspects of distributed computing technology, such as grids and clouds. We also regularly feature articles on distributed computing-enabled research in a large variety of disciplines, including physics, biology, sociology, earth sciences, archaeology, medicine, disaster management, crime, and art. (Note that we do not cover stories that are purely about commercial technology.)

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  • richardmitnick 1:19 pm on January 30, 2018 Permalink | Reply
    Tags: , , Cell differentiation, Cryo-electron microscopy, , , Polycomb Repressive Complex 2 (PRC2)   

    From LBNL: “Silencing Is Golden: Scientists Image Molecules Vital for Gene Regulation” 

    Berkeley Logo

    Berkeley Lab

    January 29, 2018
    Dan Krotz
    (510) 486-4019

    Structure of the human Polycomb Repressive Complex 2 (PRC2) bound to cofactors obtained by cryo-electron microscopy. Both cofactors mimic the histone protein tail to stabilize and stimulate the enzymatic activity of PRC2. (Credit: Vignesh Kasinath)

    All the trillions of cells in our body share the same genetic information and are derived from a single, fertilized egg. When this initial cell multiplies during fetal development, its daughter cells become more and more specialized. This process, called cell differentiation, gives rise to all the various cell types, such as nerve, muscle, or blood cells, which are diverse in shape and function and make up tissues and organs. How can the same genetic blueprint lead to such diversity? The answer lies in the way that genes are switched on or off during the course of development.

    Scientists at Lawrence Berkeley National Laboratory (Berkeley Lab) have been studying the molecules that act at the genetic level to give rise to different types of cells. Some of these molecules are a complex of proteins called the Polycomb Repressive Complex 2 (PRC2) that is involved in “silencing” genes so that they are not “read” by the cellular machinery that decodes genetic information, effectively keeping the genetic information in the “off” state.

    In two new studies, a team of researchers led by Eva Nogales, senior faculty scientist in the Molecular Biophysics and Integrated Bioimaging (MBIB) Division, has gained insight into the structure of PRC2 and the ways in which it is regulated to affect gene silencing. Their work was reported on January 18 in the journal Science and on January 29 in Nature Structural and Molecular Biology by Eva Nogales and postdoctoral researchers Vignesh Kasinath and Simon Poepsel.

    Both publications provide a structural framework to understand PRC2 function, and in the case of the latter, the structures are the first to illustrate how a molecule of this type engages with its substrate. The structural descriptions of human PRC2 with its natural partners in the cell lend important insight into the mechanism by which the PRC2 complex regulates gene expression. This information could provide new possibilities for the development of therapies for cancer.

    PRC2 is a gene regulator that is vital for normal development. Genomic DNA is packaged into nucleosomes, which are formed by histone proteins that have DNA wrapped around them. Histone proteins have long polypeptide tails that can be modified by the addition and removal of small chemical groups. These modifications influence the interaction of nucleosomes with each other and other protein complexes in the nucleus. The function of PRC2 in the cell is to make a particular chemical change in one of the histones. The genes in the regions of the genome that have been modified by PRC2 are switched off, or become silenced.

    This montage of the full PRC2 with two nucleosomes is based on the superposition of the cryo-EM maps of PRC2 with and without the nucleosomes to show the consistency of the observed nucleosome binding configuration with the full PRC2 structure. (Credit: Simon Poepsel)

    “Not surprisingly, elaborate mechanisms have evolved to ensure that PRC2 marks the correct regions for silencing at the right time,” said Nogales, who is also a Howard Hughes Medical Investigator and professor of Biochemistry, Biophysics and Structural Biology at the University of California, Berkeley. Failure of this regulation not only impairs the process of development, but also contributes to the reversal of cell differentiation and the uncontrolled cell growth that are the hallmarks of cancer. “Therefore,” Nogales continued, “gaining insight into how PRC2 function is adjusted both in space and time is crucial to understanding cell development.”

    Nogales and her team use structural biology to elucidate how biomolecules, particularly proteins and nucleic acids (DNA, RNA), are organized and combine to form functional biological assemblies. Obtaining detailed insights into their three-dimensional shape will not only help to understand how they function but also how this function is regulated in the cell. These two studies rely on cryo-electron microscopy for imaging the biomolecules, a technique that can see large biomolecules on a very small scale and in multiple conformations. Kasinath and Poepsel, have now solved the structure of PRC2, which provides a framework to understand how this complex is regulated to modify histone proteins.

    The first study, published January 18 in Science by Kasinath, Poepsel, Nogales, and coworkers, visualized the architecture of the complete PRC2 in atomic detail. First author Vignesh Kasinath said, “It took three years of work to obtain this high-resolution structure of all the parts, or subunits, that make up a functional PRC2, as well as visualize how additional protein subunits, called cofactors, may help regulate its activity. Remarkably, both cofactors mimic the histone protein tail in their binding to PRC2 suggesting that cofactors and histone tails together work hand-in-hand to regulate PRC2 function. This structural work holds great promise for new drug development to fight PRC2 dysfunction in cancer.”

    This work is complemented by a second study that presents snapshots of PRC2 binding to the histone proteins that it modifies as a signal for gene silencing. The structures, which have been published in Nature Structural and Molecular Biology on January 29 by Poepsel, Kasinath and Nogales this week, illustrate beautifully the action of this sophisticated complex. “PRC2 can simultaneously engage two nucleosomes,” said Poepsel, first author of this study. “Our cryo-EM images help us understand how the complex can recognize the presence of a histone modification in one nucleosome and place the same tag onto a neighboring nucleosome.” This cascade of activity enables PRC2 to spread this modification over the entire neighboring gene loci, thereby marking it for silencing. Nogales added, “The visualization of such interactions is notoriously hard. We have made an important step forward in our general understanding of how gene regulators can bind to and recognize nucleosomes.”

    PRC2 is essential to gene regulation and expression in all multicellular organisms. The findings from both studies open up tremendous possibilities for combatting cancer while simultaneously expanding our knowledge of gene regulation at a molecular level. “Because PRC2 is deregulated in cancers, it makes a good target for potential therapeutics,” said Nogales. The fundamental understanding of PRC2 arising from these studies will have broad implications in both plant and animal biology.

    This work was funded by the Howard Hughes Medical Institute and Eli Lilly. This research used cryo-electron microscopy (cryo-EM) and made use of the unique resources of the Bay Area Cryo-EM Facility. Image analysis relied on heavy computational work that was carried out at Berkeley Lab’s National Energy Research Scientific Computing Center (NERSC), a DOE Office of Science User Facility.

    NERSC Cray XC40 Cori II supercomputer

    LBL NERSC Cray XC30 Edison supercomputer

    The Genepool system is a cluster dedicated to the DOE Joint Genome Institute’s computing needs. Denovo is a smaller test system for Genepool that is primarily used by NERSC staff to test new system configurations and software.


    PDSF is a networked distributed computing cluster designed primarily to meet the detector simulation and data analysis requirements of physics, astrophysics and nuclear science collaborations.

    Vignesh Kasinath was supported by a postdoctoral fellowship from Helen Hay Whitney and Simon Poepsel was supported by the Alexander von Humboldt foundation (Germany) as a Feodor-Lynen postdoctoral fellow.

    See the full article here .

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  • richardmitnick 1:19 pm on September 13, 2017 Permalink | Reply
    Tags: , Cryo-electron microscopy, , , , PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography), , TFIIH-Transcription factor IIH   

    From LBNL: “Berkeley Lab Scientists Map Key DNA Protein Complex at Near-Atomic Resolution” 

    Berkeley Logo

    Berkeley Lab

    September 13, 2017
    Sarah Yang
    (510) 486-4575

    The cryo-EM structure of Transcription Factor II Human (TFIIH). The atomic coordinate model, colored according to the different TFIIH subunits, is shown inside the semi-transparent cryo-EM map. (Credit: Basil Greber/Berkeley Lab and UC Berkeley)

    Chalking up another success for a new imaging technology that has energized the field of structural biology, researchers at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) obtained the highest resolution map yet of a large assembly of human proteins that is critical to DNA function.

    The scientists are reporting their achievement today in an advanced online publication of the journal Nature. They used cryo-electron microscopy (cryo-EM) to resolve the 3-D structure of a protein complex called transcription factor IIH (TFIIH) at 4.4 angstroms, or near-atomic resolution. This protein complex is used to unzip the DNA double helix so that genes can be accessed and read during transcription or repair.

    “When TFIIH goes wrong, DNA repair can’t occur, and that malfunction is associated with severe cancer propensity, premature aging, and a variety of other defects,” said study principal investigator Eva Nogales, faculty scientist at Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging Division. “Using this structure, we can now begin to place mutations in context to better understand why they give rise to misbehavior in cells.”

    TFIIH’s critical role in DNA function has made it a prime target for research, but it is considered a difficult protein complex to study, especially in humans.

    How to Capture a Protein
    It takes a large store of patience and persistence to prepare specimens of human transcription factor IIH (TFIIH) for cryo-EM. Because TFIIH exists in such minute amounts in a cell, the researchers had to grow 50 liters of human cells in culture to yield a few micrograms of the purified protein.

    Human TFIIH is particularly fragile and prone to falling apart in the flash-freezing process, so researchers need to use an optimized buffer solution to help protect the protein structure.

    “These compounds that protect the proteins also work as antifreeze agents, but there’s a trade-off between protein stability and the ability to produce a transparent film of ice needed for cryo-EM,” said study lead author Basil Greber.

    Once Greber obtains a usable sample, he settles down for several days at the cryo-electron microscope at UC Berkeley’s Stanley Hall for imaging.

    “Once you have that sample inside the microscope, you keep collecting data as long as you can,” he said. “The process can take four days straight.”

    Mapping complex proteins

    “As organisms get more complex, these proteins do, too, taking on extra bits and pieces needed for regulatory functions at many different levels,” said Eva Nogales, who is also a UC Berkeley professor of molecular and cell biology and a Howard Hughes Medical Institute investigator. “The fact that we resolved this protein structure from human cells makes this even more relevant to disease research. There’s no need to extrapolate the protein’s function based upon how it works in other organisms.”

    Biomolecules such as proteins are typically imaged using X-ray crystallography, but that method requires a large amount of stable sample for the crystallization process to work. The challenge with TFIIH is that it is hard to produce and purify in large quantities, and once obtained, it may not form crystals suitable for X-ray diffraction.

    Enter cryo-EM, which can work even when sample amounts are very small. Electrons are sent through purified samples that have been flash-frozen at ultracold temperatures to prevent crystalline ice from forming.

    Cryo-EM has been around for decades, but major advances over the past five years have led to a quantum leap in the quality of high-resolution images achievable with this technique.

    “When your goal is to get resolutions down to a few angstroms, the problem is that any motion gets magnified,” said study lead author Basil Greber, a UC Berkeley postdoctoral fellow at the California Institute for Quantitative Biosciences (QB3). “At high magnifications, the slight movement of the specimen as electrons move through leads to a blurred image.”

    Making movies

    The researchers credit the explosive growth in cryo-EM to advanced detector technology that Berkeley Lab engineer Peter Denes helped develop. Instead of a single picture taken for each sample, the direct detector camera shoots multiple frames in a process akin to recording a movie. The frames are then put together to create a high-resolution image. This approach resolves the blur from sample movement. The improved images contain higher quality data, and they allow researchers to study the sample in multiple states, as they exist in the cell.

    Since shooting a movie generates far more data than a single frame, and thousands of movies are being collected during a microscopy session, the researchers needed the processing punch of supercomputers at the National Energy Research Scientific Computing Center (NERSC) at Berkeley Lab.

    NERSC Cray Cori II supercomputer

    LBL NERSC Cray XC30 Edison supercomputer

    NERSC Hopper Cray XE6 supercomputer

    The output from these computations was a 3-D map that required further interpretation.

    “When we began the data processing, we had 1.5 million images of individual molecules to sort through,” said Greber. “We needed to select particles that are representative of an intact complex. After 300,000 CPU hours at NERSC, we ended up with 120,000 images of individual particles that were used to compute the 3-D map of the protein.”

    To obtain an atomic model of the protein complex based on this 3-D map, the researchers used PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography), a software program whose development is led by Paul Adams, director of Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging Division and a co-author of this study.

    Not only does this structure improve basic understanding of DNA repair, the information could be used to help visualize how specific molecules are binding to target proteins in drug development.

    “In studying the physics and chemistry of these biological molecules, we’re often able to determine what they do, but how they do it is unclear,” said Nogales. “This work is a prime example of what structural biologists do. We establish the framework for understanding how the molecules function. And with that information, researchers can develop finely targeted therapies with more predictive power.”

    Other co-authors on this study are Pavel Afonine and Thi Hoang Duong Nguyen, both of whom have joint appointments at Berkeley Lab and UC Berkeley; and Jie Fang, a researcher at the Howard Hughes Medical Institute.

    NERSC is a DOE Office of Science User Facility located at Berkeley Lab. In addition to NERSC, the researchers used the Lawrencium computing cluster at Berkeley Lab. This work was funded by the National Institute of General Medical Sciences and the Swiss National Science Foundation.

    See the full article here .

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  • richardmitnick 11:57 am on July 4, 2017 Permalink | Reply
    Tags: , , , Cryo-electron microscopy, , , Tilting the sample gave a more complete dataset   

    From Salk: “Tilted microscopy technique better reveals protein structures” 

    Salk Institute bloc

    Salk Institute for Biological Studies

    July 3, 2017
    No writer credit found

    Cryo-Electron Microscope. No image credit.

    Salk Institute researcher describes new cryo-EM method to facilitate a better understanding of proteins involved in disease.

    The conventional way of placing protein samples under an electron microscope during cryo-EM experiments may fall flat when it comes to getting the best picture of a protein’s structure. In some cases, tilting a sheet of frozen proteins—by anywhere from 10 to 50 degrees—as it lies under the microscope, gives higher quality data and could lead to a better understanding of a variety of diseases, according to new research led by Salk scientist Dmitry Lyumkis.

    Dmitry Lyumkis. Credit: Salk Institute.

    “People have tried to implement tilting before, but there have been a lot of challenges,” says Lyumkis, a Helmsley-Salk Fellow at the Salk Institute and senior author of the new work, published July 3, 2017 in Nature Methods. “We’ve eliminated many of these problems with our new approach.”

    Cryo-EM, or cryo-electron microscopy, is a form of transmission electron microscopy in which samples are quickly cooled to below freezing before being imaged under the microscope. Unlike other methods commonly used to determine the structure of proteins, cryo-EM lets proteins remain in their natural conformations for imaging, which could reveal new information about the structures. Understanding proteins’ structures is a vital step to developing new therapies for disease, such as in the case of HIV.

    Researchers have long assumed that proteins adopt random conformations throughout the frozen grid that’s prepared for cryo-EM experiments, which means that by taking enough images, researchers can put together a full, 3D picture of the protein’s shape(s) from all imaging directions. But for many proteins, the approach seems to fall short, and parts of the proteins’ structures remain missing.

    “Researchers are starting to think that the proteins on a cryo-EM grid don’t adopt random conformations after all, but rather stick to the top or bottom of the sample grid in preferred orientations,” says Lyumkis. “Thus we may not be getting the full picture of proteins’ structures. More importantly, this behavior can prohibit structure determination altogether for select protein samples.”

    To understand the problem, imagine trying to look at the shadows of a dozen tin cans to figure out their shape but seeing only circles because all the cans are exactly upright. By making the light—or electron beam, in the case of cryo-EM—hit the samples at an angle, though, you’d be able to see the true shape better.

    When researchers have tried to tilt samples under a microscope in the past, they’ve been limited by poor resolution: an angle means that the electron beam has to travel through a thicker grid. Samples are also more likely to move within the frozen grid when they’re tilted, blurring out the data. And technically, analyzing data from a tilted sample is also more challenging, since cryo-EM methods were designed with the assumption that the grid containing proteins was always at the same distance from the microscope.

    To tackle these challenges, Lyumkis and his colleagues changed the materials used to create the cryo-EM grid, recorded movies of their data rather than still images, and developed new computational methods to analyze the information.

    When they tested the new approach on the influenza hemagglutinin protein, a notoriously hard protein to characterize using cryo-EM, the team found that tilting the sample gave a more complete dataset. When the protein sample was flat, typical algorithms introduced false positive shape to the protein that wasn’t backed up by experimental data. That wasn’t the case when it was tilted.

    “Due to the geometry of the data collection when we tilt, we fill up much more data characterizing the molecules, giving us a more complete picture of the protein’s shape” says Lyumkis.

    The algorithms that Lyumkis and his team developed—which include ways to analyze whether a cryo-EM experiment is introducing bad data, as well as the methods to interpret a tilted experiment—are now openly available. They hope other researchers will start using them and that it becomes a standard metric for cryo-EM structure validation (since most experimentally derived structures suffer from missing information to different extents).

    “One of the ideas we’re looking at now is whether data collection should always be performed at a tilt rather than in the conventional way,” says Lyumkis. “It won’t hurt and it should help.”

    Other researchers on the study were Yong Zi Tan, Philip Baldwin, Clinton Potter and Bridget Carragher of the New York Structural Biology Center, and Joseph David and James Williamson of The Scripps Research Institute.

    The work and the researchers involved were supported by grants from the Agency for Science, Technology, and Research Singapore, the Leona M. and Harry B. Helmsley Charitable Trust, the U.S. National Institutes of Health, the Jane Coffin Childs Foundation, the National Institute of Aging, the National Institute of General Medical Sciences and the Simons Foundation.

    See the full article here .

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    Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

  • richardmitnick 12:39 pm on January 23, 2017 Permalink | Reply
    Tags: , Cryo-electron microscopy, Harvard Cryo-Electron Microscopy Center for Structural Biology, ,   

    From HMS: “The Future of Molecular Visualization” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    January 18, 2017
    No writer credit found

    New cryo-electron microscopy center to transform biomedical imaging

    Cryo-EM images can reveal new insights into how the molecular machines of a cell operate. Image: Maofu Liao.

    Seeing a molecule in a microscope was once the stuff of science fiction. No longer.

    With the creation of the Harvard Cryo-Electron Microscopy Center for Structural Biology in the Longwood Medical Area, Harvard University today launched a pivotal initiative in molecular visualization, which promises remarkable advances in scientists’ ability to see molecules directly.

    Visualizing molecules at the level of atoms enables in-depth understanding of molecular mechanisms in both normal and disease states. Seeing subtle molecular details will fuel the development of next-generation precision therapeutics.

    The new center emerged from a bold and visionary collaboration among partners from Harvard Medical School, the University’s Office of the Provost, Boston Children’s Hospital and Dana-Farber Cancer Institute.

    “This new center demonstrates how Harvard and its affiliated institutions can partner to establish leading-edge facilities and resources that accelerate biomedical discoveries,” said Alan Garber, provost of Harvard University.

    Stephen Blacklow, the Gustavus Adolphus Pfeiffer Professor and chair of the Department of Biological Chemistry and Molecular Pharmacology at HMS, remarked, “The cooperation and resolve shown by all participants in pursuit of this effort has been truly impressive and foreshadows an outstanding future for molecular visualization at Harvard.”

    George Q. Daley, dean of HMS, said, “We now have a microscope that allows us to see single molecules at the atomic level. This innovation will energize science in the hospitals and on the Quad, catalyzing translational research to see where it can bear on disease.”

    “Cryo-electron microscopy is an important tool to reveal the structures of many building blocks essential to our understanding of human biology and the alterations that affect health and disease states,” added Barbara J. McNeil, former acting dean of HMS.

    “We are extremely excited about the new HMS center and look forward over the coming years to an explosion in our understanding of cellular machines,” said Wade Harper, the Bert and Natalie Vallee Professor of Molecular Pathology and chair of the Department of Cell Biology at HMS.

    Cryo-electron microscopy (cryo-EM) represents the latest frontier in imaging deployed by structural biologists.

    See the full article here .

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    Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

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    Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

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  • richardmitnick 2:49 pm on January 3, 2017 Permalink | Reply
    Tags: A day in the life of a molecular machine, , Cryo-electron microscopy, Georgia State University, , , ,   

    From Science Node: “A day in the life of a molecular machine” 

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    Science Node

    01 Dec, 2016 [Where has this been?]
    Jorge Salazar

    Courtesy Macmillan Publishers Ltd; Yuan He, et al.

    Supercomputers and cryo-electron microscopy take a perfect picture of molecular machines.

    It sounds like something out of Star Trek: Nano-sized robots self-assemble to form biological machines that do the work of living. And yet this is not science fiction – this really happens.

    Every cell in our body has identical DNA, the twisted staircase of nucleic acids uniquely coded to each organism. Molecular machines take pieces of DNA called genes and make a brain cell when needed, instead of, say, a bone cell.

    Model scientist. Ivaylo Ivanov, associate professor of chemistry at Georgia State University, conducted over four million hours of supercomputer simulations to model molecular machines.

    Scientists today are just starting to understand their structure and function using the latest microscopes and supercomputers.

    Cryo-electron microscopy (cryo-EM) combined with supercomputer simulations have created the best model yet of a vital molecular machine, the human pre-initiation complex (PIC).

    “For the first time, structures have been detailed of the complex groups of molecules that open human DNA,” says study co-author Ivaylo Ivanov, associate professor of chemistry at Georgia State University.

    Ivanov led the computational work that modeled the atoms of the different proteins that act like cogs of the PIC molecular machine.

    The experiment began with images painstakingly taken of PIC. They were made by a group led by study co-author Eva Nogales, senior faculty scientist at Lawrence Berkeley National Laboratory.

    Nogales’ group used cryo-EM to freeze human PIC bound to DNA before zapping it with electron beams. Thanks to recent advances, cryo-EM can now image at near atomic resolution large and complicated biological structures that have proven too difficult to crystalize.

    In all, over 1.4 million cryo-EM ‘freeze frames’ of PIC were processed using supercomputers at the National Energy Research for Scientific Computing Center (NERSC)

    NERSC CRAY Cori supercomputer
    NERSC CRAY Cori supercomputer
    LBL NERSC Cray XC30 Edison supercomputer
    LBL NERSC Cray XC30 Edison supercomputer

    “Cryo-EM is going through a great expansion,” Nogales says. “It is allowing us to get higher resolution of more structures in different states so that we can describe several pictures showing how they are moving. We don’t see a continuum, but we see snapshots through the process of action.”

    Using eXtreme Science and Engineering Discovery Environment (XSEDE) resources, scientists next built an accurate model that made physical sense of the density maps of PIC.

    Ice queen. Eva Nogales, senior faculty scientist at the Lawrence Berkeley National Laboratory uses cryo-electron microscopy to produce near atomic-level resolution images of molecular structure. Courtesy Eva Nogales.

    To model complex molecular machines, including those for this study, Ivanov’s team ran over four million core hours of simulations on the Stampede supercomputer at the Texas Advanced Computing Center (TACC).
    TACC bloc
    Dell Poweredge U Texas Austin Stampede Supercomputer. Texas Advanced Computer Center 9.6 PF
    “Dell Poweredge U Texas Austin Stampede Supercomputer. Texas Advanced Computer Center 9.6 PF

    The goal of all this computational effort is to produce atomic models that tell the full story of the structure and function of the protein complex of molecules. To get there, Ivanov’s team took the twelve components of the PIC assembly and created homology models for each component that accounted for their amino acid sequences and their relation to similar known protein 3-D structures.

    XSEDE was “absolutely necessary” for this modeling, says Ivanov. “When we include water and counter ions in addition to the PIC complex in a molecular dynamics simulation box, we get the simulation system size of over a million atoms. For that we need to go to a thousand cores. In this case, we went up to two thousand and forty-eight cores – for that we needed Stampede,” Ivanov said.

    One of the insights gained in the study is a working model of how PIC opens the otherwise stable DNA double helix for transcription. Imagine a cord made of two threads twisted around each other, Nogales explains. Hold one end very tightly, then grab the other and twist it in the opposite direction of the threading to unravel the cord. That’s basically how the living machines that keep us alive do it.

    Changing stations. By aligning the three models of holo-PICs, sequential states are morphed with a special focus on the nucleic acids regions. Courtesy Macmillan Publishers Ltd; Yuan He, et al.

    Both scientists said that they are just beginning to get an atomic-level understanding of transcription, crucial to gene expression and ultimately disease.

    “Many disease states come about because there are errors in how much a certain gene is being read and how much a certain protein with a certain activity in the cell is present,” Nogales says. “Those disease states could be due to excess production of the protein, or conversely not enough. It is very important to understand the molecular process that regulates this production so that we can understand the disease state.”

    While this fundamental work does not directly produce cures, it does lay the foundation to help develop them in the future, said Ivanov. “In order to understand disease, we have to understand how these complexes function in the first place… A collaboration between computational modelers and experimental structural biologists could be very fruitful in the future. ”

    The results,Near-atomic resolution visualization of human transcription promoter opening, were recently published in Nature.

    The article was authored by Yuan He, Lawrence Berkeley National Laboratory and now at Northwestern University; Chunli Yan and Ivaylo Ivanov, Georgia State University; Jie Fang, Carla Inouye, Robert Tjian, Eva Nogales, UC Berkeley.

    Funding came from the National Institute of General Medical Sciences (NIH) and the National Science Foundation.

    See the full article here .

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  • richardmitnick 7:57 am on July 23, 2016 Permalink | Reply
    Tags: , Cryo-electron microscopy,   

    From Penn State: “Super-Cold Microscope Has Super Cool Uses” 

    Penn State Bloc

    Pennsylvania State University

    July 22, 2016
    No writer credit found

    Cryo-electron microscopes enable the creation of detailed atomic and molecular models such as this one, a full cryo-EM reconstruction of F-actin and a corresponding close-up view with the atomic and molecular model of an F-actin subunit (cyan) and tropomyosin (yellow).
    Image: Courtesy of Julian von der Ecken and Prof. Dr. Stefan Raunser, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Cryo-EM image of human papillomavirus. These images can be used to reconstruct the structure of the virus in 3-D. This approach also works for proteins, protein complexes, DNA-complexes. Resulting maps can be resolved at atomic resolution. Image: Susan Hafenstein, Director of Cryo-EM Imaging Facility, Penn State College of Medicine

    Cryo-electron microscope at Penn State

    While “go big” is the motto for many science initiatives, Penn State researchers are hoping a cutting-edge microscope will allow them to “go deep” to promote biomedical research and discoveries in materials science.

    The purchase of a cryo-electron microscope, recently approved by the Penn State Board of Trustees, freezes samples at cryogenic temperatures — which usually start at or below -238 degree Fahrenheit — to cut down on radioactive interference and improve resolution to the atomic level. Penn State’s cryo-electron microscope will allow researchers to see down to 3 Angstroms, almost at the resolution of a single carbon atom, according to Jim Marden, professor of biology and director of operations, Huck Institutes of the Life Sciences.

    Marden added that the device will likely find immediate use in research conducted by Penn State scientists on enzymes, viruses and the structure of RNA. Solving these structures will lead to other discoveries and new disease treatments, since drug and vaccine design require knowledge about how molecules interact at the scale of individual atoms. But, its potential to lead to other discoveries and treatments is considerable.

    “Now we have a novel freezing technique, image sensor and algorithms that allow us to see these detailed structures and thus understand function,” said Marden. “We are on the edge of revealing how life works and we know that this involves imaging — and being able to see detail.”

    The cryo-electron microscope is a specialized piece of research equipment that costs $8.6 million. Marden expects the Materials Characterization Lab in the Institute for Materials Research to also benefit from the cryo-electron microscope.

    “The materials scientists will use this scope to better understand soft materials and this new microscope will allow new types of collaborations at the interface of the fields of materials science and life sciences,” said Marden. “For example, targeted delivery of drugs using nanoparticles will benefit greatly from visualizing the engineered particles and how they interact with cell surfaces.

    The microscope will also serve as a critical tool in reaffirming Penn State’s role as a leader in promoting the convergence of the life sciences and materials science, according to Peter Hudson, the Willaman Professor of Biology and director of the Huck Institutes of Life Sciences.

    “We are at a most exciting time in the life sciences — the technological developments with the Cryo EM will allow us to interpret function from structure and in so doing revolutionize biomedical, food, disease and materials research,” said Hudson. “Universities are at an auspicious junction and the leading universities like Penn State need to invest in this technology to be able to be in the leading pack of researchers and entrepreneurs.”

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  • richardmitnick 2:03 pm on September 13, 2015 Permalink | Reply
    Tags: , Cryo-electron microscopy,   

    From Nature: “The revolution will not be crystallized: a new method sweeps through structural biology” 

    Nature Mag

    09 September 2015
    Ewen Callaway

    Move over X-ray crystallography. Cryo-electron microscopy is kicking up a storm by revealing the hidden machinery of the cell.

    Illustration by Viktor Koen

    In a basement room, deep in the bowels of a steel-clad building in Cambridge, a major insurgency is under way.

    A hulking metal box, some three metres tall, is quietly beaming terabytes’ worth of data through thick orange cables that disappear off through the ceiling. It is one of the world’s most advanced cryo-electron microscopes: a device that uses electron beams to photograph frozen biological molecules and lay bare their molecular shapes.

    Cryo-Electron Microscope
    Cryo-Electron Microscope

    The microscope is so sensitive that a shout can ruin an experiment, says Sjors Scheres, a structural biologist at the UK Medical Research Council Laboratory of Molecular Biology (LMB), as he stands dwarfed beside the £5-million (US$7.7-million) piece of equipment. “The UK needs many more of these, because there’s going to be a boom,” he predicts.

    In labs around the world, cryo-electron microscopes such as this one are sending tremors through the field of structural biology. In the past three years, they have revealed exquisite details of protein-making ribosomes, quivering membrane proteins and other key cell molecules, discoveries that leading journals are publishing at a rapid clip. Structural biologists say — without hyperbole — that their field is in the midst of a revolution: cryo-electron microscopy (cryo-EM) can quickly create high-resolution models of molecules that have resisted X-ray crystallography and other approaches, and labs that won Nobel prizes on the back of earlier techniques are racing to learn this upstart method. The new models reveal precisely how the essential machinery of the cell operates and how molecules involved in disease might be targeted with drugs.

    “There’s a huge range of very important biological problems that are now open to being tackled in a way that they could never before,” says David Agard, a structural cell biologist at the University of California, San Francisco.

    Scheres was recruited to the LMB several years ago to help push cryo-EM technology to its limits — and he and his colleagues have done just that. Last month, they reported one of the burgeoning field’s most impressive feats: a startlingly clear picture of an enzyme implicated in Alzheimer’s disease, showing the position of its 1,200 or so amino acids down to a resolution of a few tenths of a nanometre1.

    Biologists are now pushing the technique further to deduce ever more detailed structures of small and shape-shifting molecules — a challenge even for cryo-EM. “Whether you call it revolution or a quantum leap, the fact is that the gates have opened,” says Eva Nogales, a structural biologist at the University of California, Berkeley.

    Crystal coaxing

    Spend a bit of time with a structural biologist and they will probably mention their field’s unofficial motto: ‘structure is function’. Only by knowing the atom-by-atom arrangement of a biomolecule can researchers grasp how it works — how, for instance, the ribosome reads strands of messenger RNA to manufacture proteins, or how molecular pores flip open and shut. For decades, one technique enjoyed a near monopoly in elucidating protein structures to this level of detail: X-ray crystallography, in which scientists persuade proteins to form into crystals, then blast X-rays at them and decipher the protein’s structure from patterns that the X-rays make when they bounce off (see ‘Structure solvers’). Of the more than 100,000 entries in the Protein Data Bank, a popular repository of protein structures, about 90% were solved by this technique. It has contributed to more than a dozen Nobel prizes, including the one awarded in 1962 for revealing DNA’s double helix.

    X-ray image: SPL

    But although X-ray crystallography has been structural biologists’ best tool, it also has major limitations. It can take researchers years to find ways of forming some recalcitrant proteins into large crystals that are suitable for analysis, and many fundamentally important molecules — such as proteins that are embedded in cell membranes or that make up complex molecular machines — have defied crystallization.

    X-ray crystallography was certainly king when biologist Richard Henderson arrived at the LMB in 1973 to study a protein called bacteriorhodopsin, which uses light energy to pump protons across a membrane. Henderson and his colleague Nigel Unwin had managed to make two-dimensional crystals from the protein, but they were unsuitable for X-ray diffraction. So the pair decided to try electron microscopy instead.

    At the time, electron microscopy was used to study viruses or slices of tissue that had been treated with heavy-metal stains. A beam of electrons is fired at a sample, and the emerging electrons are detected and used to map out the structure of the materials they smashed into. This approach produced the first detailed image of a virus — a tobacco pathogen — but the stain made it difficult to see individual proteins, let alone the atomic details that the X-rays were revealing. “It was blobby stuff or negative-stained, and you would see outlines of molecules,” says Agard.

    In a pivotal step, Henderson and Unwin omitted the stain when they used electron microscopy to image crystal sheets of bacteriorhodopsin — instead, they placed the crystals on metallic grids to make the protein stand out. “You were looking at the atoms in the protein,” says Henderson, who, with Unwin, published2 the structure of bacteriorhodopsin in 1975. “That was such a huge step forward,” Agard says. “That said, ‘OK, it will be possible to solve protein structures by EM’.”

    The cryo-EM field developed through the 1980s and 1990s; a key advance was the use of liquid ethane to flash-freeze proteins in solution and hold them still3, which is how the ‘cryo’ came to cryo-EM. But still the technique could generally resolve structures only to more than 10 Ångströms (1 Å is one-tenth of a nanometre) — nothing to rival the better than 4-Å models of X-ray crystallography, and nowhere near what was needed to use the structures for drug design. While funders such as the US National Institutes of Health were ploughing hundreds of millions of dollars into ambitious crystallography initiatives, support for cryo-EM lagged far behind.

    In 1997, when Henderson attended the annual Gordon Research Conference on 3D electron microscopy, a colleague opened the meeting with a provocative statement: cryo-EM was a “niche” method, he said, unlikely to ever supplant X-ray crystallography. But Henderson could see a different future, and he fired back a salvo in the next talk. “I said we should go for global domination of cryo-EM over all the structural methods,” he recalls.

    The revolution starts here

    In the years that followed, Henderson, Agard and other cryo-EM evangelists worked methodically on technical improvements to electron microscopes — in particular, on better ways to sense electrons. Long after digital cameras had taken the world by storm, many electron microscopists still preferred old-fashioned film because it recorded electrons more efficiently than did digital sensors. But, working with microscope manufacturers, the researchers developed a new generation of ‘direct electron detectors’ that vastly outperforms both film and digital-camera detectors.

    Available since about 2012, the detectors can capture quick-fire images of an individual molecule at dozens of frames per second. Researchers such as Scheres, meanwhile, have written sophisticated software programs to morph thousands of 2D images into sharp 3D models that, in many cases, match the quality of those deciphered with crystallography.

    Cryo-EM is suited to large, stable molecules that can withstand electron bombardment without jiggling around — so molecular machines, often built from dozens of proteins, are good targets. None has proved more suitable than ribosomes, which are braced by rigid twists of RNA. The solution of ribosome structures by X-ray crystallography won three chemists the 2009 Nobel Prize in Chemistry — but those efforts took decades. In the past couple of years, ‘ribosomania’ has gripped cryo-EM researchers, and various teams have quickly determined and published dozens of cryo-EM structures of ribosomes from a multitude of organisms, including the first high-resolution models of human ribosomes4, 5. X-ray crystallography has largely fallen by the wayside in the LMB laboratory of Venki Ramakrishnan, who shared the 2009 Nobel. For large molecules, “it’s safe to predict that cryo-EM will largely supersede crystallography”, he says.

    The rocketing number of cryo-EM publications suggests this to be true: in 2015 alone, the technique has so far been used to map the structures of more than 100 molecules. And, unlike X-ray crystallography, in which crystals lock proteins in a single, static pose, researchers can use cryo-EM to calculate the structure of a protein that has been flash-frozen in several conformations and so deduce the mechanisms by which it works.

    Advances in cryo-electron microscopy have helped scientists to produce valuable models of a string of proteins. This year, two teams revealed the first high-resolution structures of ribosomes, which are built from dozens of protein and RNA molecules, at resolutions of 3.5 Ångströms and 3.6 Å. (An Ångström is one ten-billionth of a metre.)

    The TRPV1 channel detects the burn of chilli peppers, and this 3.4-Å structure is considered super-hot in the structural-biology world. Like many other proteins that are sandwiched into the cellular membrane, TRPV1 had been impossible to solve by X-ray crystallization. Its structure could guide the design of new painkillers.

    The enzyme γ-secretase makes the toxic amyloid-β molecules implicated in Alzheimer’s disease. It is a challenge for cryo-EM, which struggles with such small, floppy and asymmetrical proteins. So an atomic structure published this year at 3.4 Å resolution is considered a triumph, and could help in the search for disease therapies.

    This 2.2-Å structure of a bacterial enzyme, β-galactosidase, that snips lactose sugar into simpler molecules, is the sharpest cryo-EM image on record. At that resolution, water molecules (here shown in yellow within part of the structure) and ions associated with the protein come into view, and drug designers can see how they might alter a protein’s activity.

    Atomic structures can reveal how proteins work. Earlier this year, a team worked out the structure of three confirmations of a yeast enzyme known as vacuolar ATPase that burns ATP to power a membrane proton pump. They used these to create a ‘molecular movie’ that showed the pump’s action.

    Researchers have used X-ray crystallography to study how anthrax bacteria deliver killer toxins into cells through a molecular pore, but an image of the open pore has been elusive. At last, this year a team used cryo-EM to come up with a 2.9-Å model of the pore.

    In May, structural biologist John Rubinstein at the University of Toronto, Canada, and his colleagues used around 100,000 cryo-EM images to create a ‘molecular movie’ of a rotor-shaped enzyme called V-ATPase, which pumps protons in and out of cell vacuoles by burning ATP6. “What we saw is that everything is flexible,” Rubinstein says. “It’s bending and twisting and deforming.” He thinks that the enzyme’s flexibility helps it to efficiently transmit energy released by ATP to the pump.

    And when a team led by Nogales in 2013 pieced together cryo-EM images of a complex that orchestrates the transcription of DNA into RNA, they discovered that an entire arm swings 100 Å around the DNA strand like a crane, potentially influencing whether a gene is transcribed7. “I think this is beautiful,” says Nogales. “It’s a true insight into how these biological machines work.”

    Small and beautiful

    Now that cryo-EM has hit its stride, experts are looking for grander challenges. For many, the most coveted targets are smaller proteins sandwiched in cellular membranes. These tend to be linchpins in cellular signalling pathways, as well as popular drug targets. They are also notoriously difficult to crystallize, and imaging individual proteins with cryo-EM is tough because it is harder to extract the signal from the background noise.

    These hurdles did not stop Yifan Cheng, a biophysicist at the University of California, San Francisco (UCSF), from attempting cryo-EM on a small membrane protein called TRPV1, which detects the molecule that gives chilli peppers their burn and is closely related to other pain-sensing proteins. A team led by his collaborator David Julius, a UCSF physiologist, had failed to crystallize the protein. The cryo-EM project was slow-going at first, but the same technical advances that drove ribosomania produced a 3.4-Å structure of TRPV1 in late 2013. The report8 was a thunderbolt to the field, because it showed that cryo-EM could conquer small, medically important molecules. “I literally lost an entire night’s sleep when I saw that,” says Rubinstein.

    More sleepless nights are likely to follow. “There’s going to be a huge explosion in the number of membrane-protein structures that get solved,” says Agard.

    One such solution was that published last month1 by Scheres, structural biologist Yigong Shi of Tsinghua University in Beijing and their team. They produced a model of γ-secretase — a protein that makes the amyloid-β molecule that is linked to Alzheimer’s disease. The 3.4-Å-resolution map reveals that γ-secretase mutations that cause rare inherited forms of Alzheimer’s map to two ‘hotspots’ in the enzyme and seem to influence its ability to form toxic amyloid-β particles. The structure could help researchers to understand why drugs that inhibit the enzyme have failed in past clinical trials, and help them to design new pharmaceuticals. “Stunning” is how Cheng describes the structure.

    Results such as these are attracting the attention of drug companies hoping to study medically important proteins that have resisted crystallography. Scheres is working with New York-based pharmaceutical giant Pfizer on ion channels, a broad class of membrane protein that includes pain-sensing molecules and neurotransmitter receptors. “I’ve been contacted by almost everybody,” says Nogales of the drug companies lining up at her door.

    But despite the advances, many in the field see room for further improvement. They hope to devise better electron detectors and better methods for preparing protein samples. This would allow scientists to image proteins that are even smaller and more dynamic, and at even greater resolution than before. A 2.2-Å structure of a bacterial enzyme published in May9 showed just how sharp cryo-EM structures can get.

    Like any burgeoning field, this one has growing pains. Some experts worry that researchers rushing to use the technique could produce problematic results. A 2013 structure of an HIV surface protein10 was questioned by scientists who said that the images used to build the model were white noise11. Since then, X-ray and cryo-EM models generated by other teams have challenged the original model, but the researchers have stood by their result12. This June, at the field’s Gordon conference, researchers wanting more quality control passed a resolution urging journals to provide referees with details of how cryo-EM structures were created.

    Costs could slow the spread of the technology. Scheres estimates that the LMB spends around £3,000 per day running its cryo-EM facility, plus another £1,000 on electricity, most of it for computers needed to store and process the images. “You’re £4,000 per day lighter if you want to do this. That, for many places, is a very high cost,” he says. To make cryo-EM more accessible, some funders have established shared facilities at which researchers can book time. The Howard Hughes Medical Institute (HHMI) operates a cryo-EM lab on its Janelia Farm Campus in Virginia that is open to HHMI-funded investigators based elsewhere. In the United Kingdom, a national cryo-EM facility funded by the government and the Wellcome Trust opened this year in Didcot, near Oxford. “There is a real tidal wave of people wanting to learn about it,” says Helen Saibil, a structural biologist at Birkbeck, University of London, who helped to establish the UK facility.

    Riding the wave is Rod MacKinnon, a biophysicist at Rockefeller University in New York City, who shared the 2003 Nobel Prize in Chemistry for determining the crystal structure of certain ion channels, but who is now deep into cryo-EM. “I’m on a steep slope of a learning curve, which always thrills me,” says MacKinnon, who hopes to use the method to study how ion channels open and close.

    Henderson’s tongue may have been firmly in his cheek when he declared back in 1997 that cryo-EM could rule the structural-biology world. But nearly 20 years later, his prediction is looking less like hyperbole than it did then. “If it carries on, and all the technical problems are solved, cryo-EM could indeed become, not just a first choice, but a dominant technology,” he says. “We are probably halfway there.”

    See the full article for reference list.

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