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  • richardmitnick 5:53 pm on November 12, 2018 Permalink | Reply
    Tags: , , , MicroED-micro-electron diffraction, , NMR-nuclear magnetic resonance, , , X-ray crystallography,   

    From Caltech: “From Beaker to Solved 3-D Structure in Minutes” 

    Caltech Logo

    From Caltech


    Whitney Clavin
    (626) 395-1856

    Graduate student Tyler Fulton prepares samples of small molecules in a lab at Caltech. Credit: Caltech

    Close-up of a powder containing small molecules like those that gave rise to 3-D structures in the new study. (The copper piece is a sample holder used with microscopes.) Credit: Caltech/Stoltz Lab

    Brian Stoltz and Tyler Fulton. Credit: Caltech

    UCLA/Caltech team uncovers a new and simple way to learn the structures of small molecules.

    In a new study that one scientist called jaw-dropping, a joint UCLA/Caltech team has shown that it is possible to obtain the structures of small molecules, such as certain hormones and medications, in as little as 30 minutes. That’s hours and even days less than was possible before.

    The team used a technique called micro-electron diffraction (MicroED), which had been used in the past to learn the 3-D structures of larger molecules, specifically proteins. In this new study, the researchers show that the technique can be applied to small molecules, and that the process requires much less preparation time than expected. Unlike related techniques—some of which involve growing crystals the size of salt grains—this method, as the new study demonstrates, can work with run-of-the-mill starting samples, sometimes even powders scraped from the side of a beaker.

    “We took the lowest-brow samples you can get and obtained the highest-quality structures in barely any time,” says Caltech professor of chemistry Brian Stoltz, who is a co-author on the new study, published in the journal ACS Central Science. “When I first saw the results, my jaw hit the floor.” Initially released on the pre-print server Chemrxiv in mid-October, the article has been viewed more than 35,000 times.

    The reason the method works so well on small-molecule samples is that while the samples may appear to be simple powders, they actually contain tiny crystals, each roughly a billion times smaller than a speck of dust. Researchers knew about these hidden microcrystals before, but did not realize they could readily reveal the crystals’ molecular structures using MicroED. “I don’t think people realized how common these microcrystals are in the powdery samples,” says Stoltz. “This is like science fiction. I didn’t think this would happen in my lifetime—that you could see structures from powders.”

    This movie [animated in the full article] is an example of electron diffraction (MicroED) data collection, in which electrons are fired at a nanocrystal while being continuously rotated. Data from the movie are ultimately converted to a 3-D chemical structure. Credit: UCLA/Caltech

    The results have implications for chemists wishing to determine the structures of small molecules, which are defined as those weighing less than about 900 daltons. (A dalton is about the weight of a hydrogen atom.) These tiny compounds include certain chemicals found in nature, some biological substances like hormones, and a number of therapeutic drugs. Possible applications of the MicroED structure-finding methodology include drug discovery, crime lab analysis, medical testing, and more. For instance, Stoltz says, the method might be of use in testing for the latest performance-enhancing drugs in athletes, where only trace amounts of a chemical may be present.

    “The slowest step in making new molecules is determining the structure of the product. That may no longer be the case, as this technique promises to revolutionize organic chemistry,” says Robert Grubbs, Caltech’s Victor and Elizabeth Atkins Professor of Chemistry and a winner of the 2005 Nobel Prize in Chemistry, who was not involved in the research. “The last big break in structure determination before this was nuclear magnetic resonance spectroscopy, which was introduced by Jack Roberts at Caltech in the late ’60s.”

    Like other synthetic chemists, Stoltz and his team spend their time trying to figure out how to assemble chemicals in the lab from basic starting materials. Their lab focuses on such natural small molecules as the fungus-derived beta-lactam family of compounds, which are related to penicillins. To build these chemicals, they need to determine the structures of the molecules in their reactions—both the intermediate molecules and the final products—to see if they are on the right track.

    One technique for doing so is X-ray crystallography, in which a chemical sample is hit with X-rays that diffract off its atoms; the pattern of those diffracting X-rays reveals the 3-D structure of the targeted chemical. Often, this method is used to solve the structures of really big molecules, such as complex membrane proteins, but it can also be applied to small molecules. The challenge is that to perform this method a chemist must create good-sized chunks of crystal from a sample, which isn’t always easy. “I spent months once trying to get the right crystals for one of my samples,” says Stoltz.

    Another reliable method is NMR (nuclear magnetic resonance), which doesn’t require crystals but does require a relatively large amount of a sample, which can be hard to amass. Also, NMR provides only indirect structural information.

    Before now, MicroED—which is similar to X-ray crystallography but uses electrons instead of X-rays—was mainly used on crystallized proteins and not on small molecules. Co-author Tamir Gonen, an electron crystallography expert at UCLA who began developing the MicroED technique for proteins while at the Howard Hughes Medical Institute in Virginia, said that he only started thinking about using the method on small molecules after moving to UCLA and teaming up with Caltech.

    “Tamir had been using this technique on proteins, and just happened to mention that they can sometimes get it to work using only powdery samples of proteins,” says Hosea Nelson (PhD ’13), an assistant professor of chemistry and biochemistry at UCLA. “My mind was blown by this, that you didn’t have to grow crystals, and that’s around the time that the team started to realize that we could apply this method to a whole new class of molecules with wide-reaching implications for all types of chemistry.”

    The team tested several samples of varying qualities, without ever attempting to crystallize them, and were able to determine their structures thanks to the samples’ ample microcrystals. They succeeded in getting structures for ground-up samples of the brand-name drugs Tylenol and Advil, and they were able to identify distinct structures from a powdered mixture of four chemicals.

    The UCLA/Caltech team says it hopes this method will become routine in chemistry labs in the future.

    “In our labs, we have students and postdocs making totally new and unique molecular entities every day,” says Stoltz. “Now we have the power to rapidly figure out what they are. This is going to change synthetic chemistry.”

    The study was funded by the National Science Foundation, the National Institutes of Health, the Department of Energy, a Beckman Young Investigators award, a Searle Scholars award, a Pew Scholars award, the Packard Foundation, the Sloan Foundation, the Pew Charitable Trusts, and the Howard Hughes Medical Institute. Other co-authors include Christopher Jones,Michael Martynowycz, Johan Hattne, and Jose Rodriguez of UCLA; and Tyler Fulton of Caltech.

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    The California Institute of Technology (commonly referred to as Caltech) is a private research university located in Pasadena, California, United States. Caltech has six academic divisions with strong emphases on science and engineering. Its 124-acre (50 ha) primary campus is located approximately 11 mi (18 km) northeast of downtown Los Angeles. “The mission of the California Institute of Technology is to expand human knowledge and benefit society through research integrated with education. We investigate the most challenging, fundamental problems in science and technology in a singularly collegial, interdisciplinary atmosphere, while educating outstanding students to become creative members of society.”

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  • richardmitnick 1:50 pm on November 7, 2018 Permalink | Reply
    Tags: , , , , , , , Researchers create most complete high-res atomic movie of photosynthesis to date, , X-ray crystallography,   

    From SLAC National Accelerator Lab: “Researchers create most complete high-res atomic movie of photosynthesis to date” 

    From SLAC National Accelerator Lab

    November 7, 2018

    Andrew Gordon
    (650) 926-2282

    In a major step forward, SLAC’s X-ray laser captures all four stable states of the process that produces the oxygen we breathe, as well as fleeting steps in between. The work opens doors to understanding the past and creating a greener future.

    Using SLAC’s X-ray laser, researchers have captured the most complete high-res atomic movie to date of Photosystem II, a key protein complex in plants, algae and cyanobacteria responsible for splitting water and producing the oxygen we breathe. (Gregory Stewart, SLAC National Accelerator Laboratory)

    Despite its role in shaping life as we know it, many aspects of photosynthesis remain a mystery. An international collaboration between scientists at SLAC National Accelerator Laboratory, Lawrence Berkeley National Laboratory and several other institutions is working to change that. The researchers used SLAC’s Linac Coherent Light Source (LCLS) X-ray laser to capture the most complete and highest-resolution picture to date of Photosystem II, a key protein complex in plants, algae and cyanobacteria responsible for splitting water and producing the oxygen we breathe. The results were published in Nature today.


    Explosion of life

    When Earth formed about 4.5 billion years ago, the planet’s landscape was almost nothing like what it is today. Junko Yano, one of the authors of the study and a senior scientist at Berkeley Lab, describes it as “hellish.” Meteors sizzled through a carbon dioxide-rich atmosphere and volcanoes flooded the surface with magmatic seas.

    Over the next 2.5 billion years, water vapor accumulating in the air started to rain down and form oceans where the very first life appeared in the form of single-celled organisms. But it wasn’t until one of those specks of life mutated and developed the ability to harness light from the sun and turn it into energy, releasing oxygen molecules from water in the process, that Earth started to evolve into the planet it is today. This process, oxygenic photosynthesis, is considered one of nature’s crown jewels and has remained relatively unchanged in the more than 2 billion years since it emerged.

    “This one reaction made us as we are, as the world. Molecule by molecule, the planet was slowly enriched until, about 540 million years ago, it exploded with life,” said co-author Uwe Bergmann, a distinguished staff scientist at SLAC. “When it comes to questions about where we come from, this is one of the biggest.”

    A greener future

    Photosystem II is the workhorse responsible for using sunlight to break water down into its atomic components, unlocking hydrogen and oxygen. Until recently, it had only been possible to measure pieces of this process at extremely low temperatures. In a previous paper, the researchers used a new method to observe two steps of this water-splitting cycle [Nature]at the temperature at which it occurs in nature.

    Now the team has imaged all four intermediate states of the process at natural temperature and the finest level of detail yet. They also captured, for the first time, transitional moments between two of the states, giving them a sequence of six images of the process.

    The goal of the project, said co-author Jan Kern, a scientist at Berkeley Lab, is to piece together an atomic movie using many frames from the entire process, including the elusive transient state at the end that bonds oxygen atoms from two water molecules to produce oxygen molecules.

    “Studying this system gives us an opportunity to see how metals and proteins work together and how light controls such kinds of reactions,” said Vittal Yachandra, one of the authors of the study and a senior scientist at Berkeley Lab who has been working on Photosystem II for more than 35 years. “In addition to opening a window on the past, a better understanding of Photosystem II could unlock the door to a greener future, providing us with inspiration for artificial photosynthetic systems that produce clean and renewable energy from sunlight and water.”

    Sample assembly line

    For their experiments, the researchers grow what Kern described as a “thick green slush” of cyanobacteria — the very same ancient organisms that first developed the ability to photosynthesize — in a large vat that is constantly illuminated. They then harvest the cells for their samples.

    At LCLS, the samples are zapped with ultrafast pulses of X-rays [Science] to collect both X-ray crystallography and spectroscopy data to map how electrons flow in the oxygen-evolving complex of photosystem II. In crystallography, researchers use the way a crystal sample scatters X-rays to map its structure; in spectroscopy, they excite the atoms in a material to uncover information about its chemistry. This approach, combined with a new assembly-line sample transportation system [Nature Methods], allowed the researchers to narrow down the proposed mechanisms put forward by the research community over the years.

    Mapping the process

    Previously, the researchers were able to determine the room-temperature structure of two of the states at a resolution of 2.25 angstroms; one angstrom is about the diameter of a hydrogen atom. This allowed them to see the position of the heavy metal atoms, but left some questions about the exact positions of the lighter atoms, like oxygen. In this paper, they were able to improve the resolution even further, to 2 angstroms, which enabled them to start seeing the position of lighter atoms more clearly, as well as draw a more detailed map of the chemical structure of the metal catalytic center in the complex where water is split.

    This center, called the oxygen-evolving complex, is a cluster of four manganese atoms and one calcium atom bridged with oxygen atoms. It cycles through the four stable oxidation states, S0-S3, when exposed to sunlight. On a baseball field, S0 would be the start of the game when a player on home base is ready to go to bat. S1-S3 would be players on first, second, and third. Every time a batter connects with a ball, or the complex absorbs a photon of sunlight, the player on the field advances one base. When the fourth ball is hit, the player slides into home, scoring a run or, in the case of Photosystem II, releasing breathable oxygen.

    The researchers were able to snap action shots of how the structure of the complex transformed at every base, which would not have been possible without their technique. A second set of data allowed them to map the exact position of the system in each image, confirming that they had in fact imaged the states they were aiming for.

    In photosystem II, the water-splitting center cycles through four stable states, S0-S3. On a baseball field, S0 would be the start of the game when a batter on home base is ready to hit. S1-S3 would be players waiting on first, second, and third. The center gets bumped up to the next state every time it absorbs a photon of sunlight, just like how a player on the field advances one base every time a batter connects with a ball. When the fourth ball is hit, the player slides into home, scoring a run or, in the case of Photosystem II, releasing the oxygen we breathe. (Gregory Stewart/SLAC National Accelerator Laboratory)

    Sliding into home

    But there are many other things going on throughout this process, as well as moments between states when the player is making a break for the next base, that are a bit harder to catch. One of the most significant aspects of this paper, Yano said, is that they were able to image two moments in between S2 and S3. In upcoming experiments, the researchers hope to use the same technique to image more of these in-between states, including the mad dash for home — the transient state, or S4, where two atoms of oxygen bond together — providing information about the chemistry of the reaction that is vital to mimicking this process in artificial systems.

    “The entire cycle takes nearly two milliseconds to complete,” Kern said. “Our dream is to capture 50-microsecond steps throughout the full cycle, each of them with the highest resolution possible, to create this atomic movie of the entire process.”

    Although they still have a way to go, the researchers said that these results provide a path forward, both in unveiling the mysteries of how photosynthesis works and in offering a blueprint for artificial sources of renewable energy.

    “It’s been a learning process,” said SLAC scientist and co-author Roberto Alonso-Mori. “Over the last seven years we’ve worked with our collaborators to reinvent key aspects of our techniques. We’ve been slowly chipping away at this question and these results are a big step forward.”

    In addition to SLAC and Berkeley Lab, the collaboration includes researchers from Umeå University, Uppsala University, Humboldt University of Berlin, the University of California, Berkeley, the University of California, San Francisco and the Diamond Light Source.

    Key components of this work were carried out at SLAC’s Stanford Synchrotron Radiation Lightsource (SSRL), Berkeley Lab’s Advanced Light Source (ALS) and Argonne National Laboratory’s Advanced Photon Source (APS). LCLS, SSRL, APS, and ALS are DOE Office of Science user facilities. This work was supported by the DOE Office of Science and the National Institutes of Health, among other funding agencies.




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    SLAC Campus
    SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.

  • richardmitnick 7:39 am on September 27, 2018 Permalink | Reply
    Tags: , , , BioXFEL- short for Biology with X-ray Free Electron Lasers, HWI-Hauptman-Woodward Medical Research Institute, HWI-High-Throughput Crystallization Center, UB-University at Buffalo SUNY, X-ray crystallography, , X-ray Laser technology   

    From University at Buffalo: UB, HWI and partners awarded $22.5 million to capture biology at the atomic level using X-ray lasers” 

    U Buffalo bloc.

    From University at Buffalo

    September 26, 2018
    Ellen Goldbaum
    News Content Manager
    Tel: 716-645-4605

    BioXFEL is revolutionizing bioimaging through collaborations with academia and industry, including Google Brain.

    A research consortium led by the University at Buffalo has been awarded $22.5 million from the National Science Foundation (NSF) to continue its groundbreaking work developing advanced imaging techniques for critical biological processes that are difficult, if not impossible, to see with conventional methods.

    No image credit

    BioXFEL, an NSF Science and Technology Center and UB’s first such center, was created in November 2013 with an initial, $25 million award to UB, Hauptman-Woodward Medical Research Institute (HWI) and partner institutions.

    “The successful renewal of UB’s first NSF Science and Technology Center award confirms Western New York’s leadership in the areas of X-ray crystallography and structural biology, historically based in the Hauptman-Woodward Medical Research Institute and related departments at UB, including, most recently, the Department of Materials Design and Innovation,” said Venu Govindaraju, PhD, vice president for research and economic development at UB.

    “BioXFEL center scientists have made revolutionary advances in just a few years, using X-ray lasers to probe phenomena previously hidden from view,” he said. “They have discovered about 350 new molecular structures, expanding the knowledge base by describing these structures in more than 500 publications. With these incredibly powerful new tools, they are helping us better understand some of society’s most intractable health and science problems.”

    In addition to UB and HWI, BioXFEL partners include Arizona State University, the University of Wisconsin-Milwaukee, Stanford University, Cornell University, Rice University, the University of California, San Francisco and Miami University in Ohio.

    The goal of the research is to harness the power of X-ray lasers to transform a broad range of scientific fields, focused on structural biology and drug development and extending to potential innovations in environmental technologies and the development of new materials.

    Intensely bright, incredibly short pulses

    Called BioXFEL, short for Biology with X-ray Free Electron Lasers, the consortium of UB, HWI and their partners, is dedicated to using X-ray free electron lasers, which produce incredibly intense X-rays in extremely short pulses.

    “X-ray lasers provide two huge advantages over conventional methods,” explained Edward Snell, PhD, BioXFEL director, president and CEO of HWI and professor in the Department of Materials Design and Innovation in the School of Engineering and Applied Sciences at UB. “They are intensely bright beams that allow us to see much smaller things, like nanocrystals. And their pulses are incredibly short, which allows us to see critical processes, like how drugs bind, at rates as fast as a billionth of a billionth of a second.”

    BioXFEL is developing the next-generation of X-ray-based structural biology research, a field in which Buffalo has a long and rich history. In 1985, the Nobel Prize was awarded to the late Herbert Hauptman and Jerome Karle for their work developing the groundbreaking direct methods technique, a robust means of obtaining the shape and form of pharmaceuticals and their targets that is still used today, Snell explained.

    From photograph to movie

    In the few years that BioXFEL has existed, Snell explained, its researchers have significantly expanded the detail with which biological and other processes can be imaged. “Initially, the molecular images we made were based on distinct snapshots of molecules at certain timepoints,” he said. “Now we’re going from the photograph to the movie, we’re able to see the continuous process. With this renewal, we will be able to understand the complete dynamics of biological mechanisms.”

    HWI’s role in BioXFEL stems from its high-throughput crystallization center that over the past two decades has generated 180 million images from crystallization experiments. Many of these crystals were too small to be analyzed by conventional techniques, but may be deciphered using the power of X-ray lasers.

    The same images have attracted a collaboration with Google Brain, in this case promoting the use of artificial intelligence to expedite new discoveries in protein crystallization. “Buried within all those images are clues about how to go about finding the useful data in them more easily, but there is a lot of noise and we’ve got to work out a way to tease out the clues by somehow automating the process,” he said.

    “It’s well-known that we have this archive of images at HWI generated by our High-Throughput Crystallization Center, so crystallization centers and major pharmaceutical companies worldwide have been eager to collaborate with us,” Snell said.

    Particles in solution

    UB scientist Thomas Grant, PhD, based at HWI, has used X-ray free laser techniques to develop a new way to look at molecular structures in solution, critical for understanding how proteins function in the human body. Other BioXFEL advances include:

    · Developing a method that dramatically reduces the amount of sample needed for analysis.

    · Viewing the motions of molecules during reactions called time-resolved imaging dynamics, which allowed researchers to see how antibiotic resistance develops in tuberculosis and how a virus infects its host.

    · Using X-ray lasers to probe molecular motion studies for new technologies and materials.

    · Eight supported faculty at Arizona State University, where a compact campus XFEL is under construction, who use worldwide XFEL facilities to obtain movies of molecular machines at work in photosynthesis, viruses and drugs, while developing experimental techniques and new algorithms.

    · Four supported faculty at the University of Wisconsin-Milwaukee, leading to the first movies of biological processes underlying vision, antibiotic resistance, and the extrusion of the genome from a virus.

    · New technology developed at Cornell University that has enabled the first millisecond scale mix and inject experiments: watching proteins as they work with near atomic resolution.

    The scientific work of BioXFEL takes place through collaborations between all of the partner institutions. The initial X-ray laser experiments can only be done at the Linac Coherent Light Source at BioXFEL partner Stanford University, where a mile-long facility produces a beam one-tenth of the thickness of a human hair. A handful of these facilities are opening worldwide and BioXFEL is leading research at all of them.


    BioXFEL has also implemented a diverse and vigorous set of training programs to help prepare young scientists for careers in XFEL science, including summer intern programs, graduate student support, and postdoctoral career development activities.

    BioXFEL is headquartered at 700 Ellicott St. on the Buffalo Niagara Medical Campus in the building that houses both HWI and members of the UB Department of Materials Design and Innovation.

    The NSF Science and Technology Centers: Integrative Partnerships program supports innovative, potentially transformative research and education projects that require large-scale, long-term awards. The centers foster cutting-edge research, education of the next generations of scientists and broad distribution of the knowledge and technology produced.

    See the full article here .


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    UB is a premier, research-intensive public university and a member of the Association of American Universities. As the largest, most comprehensive institution in the 64-campus State University of New York system, our research, creative activity and people positively impact the world.

  • richardmitnick 5:09 pm on June 20, 2017 Permalink | Reply
    Tags: , , In order to use the limited beam time and the precious sample material more efficiently the team developed a new method., Micro-patterned chip containing thousands of tiny pores to hold the protein crystals, , Speed up protein analysis, Structural biology, X-ray crystallography, X-ray free-electron laser,   

    From SLAC: “SLAC Experiment is First to Decipher Atomic Structure of an Intact Virus with an X-ray Laser” 

    SLAC Lab

    June 20, 2017

    Surface structure of the bovine enterovirus 2. The three virus proteins are color-coded. (Jingshan Ren/University of Oxford)

    A ground-breaking experimental method developed by an international research team will substantially speed up protein analysis.

    An international team of scientists has for the first time used an X-ray free-electron laser to unravel the structure of an intact virus particle on the atomic level. The method dramatically reduces the amount of virus material required, while also allowing the investigations to be carried out several times faster than before. This opens up entirely new research opportunities, as the research team led by Alke Meents, a scientist at Germany’s DESY lab, reports in the journal Nature Methods.

    The researchers tested their method with the Linac Coherent Light Source (LCLS) X-ray free-electron laser at the Department of Energy’s SLAC National Accelerator Laboratory. Now they are working to increase the capacity and speed of the technique in anticipation of future experiments at the European XFEL X-ray free-electron laser, which is just going into operation near Hamburg, Germany.


    European XFEL

    “This is a much-welcome and important technological development that will greatly optimize data collection at LCLS and other X-ray free-electron lasers for certain classes of challenging experiments,” says co-author Roberto Alonso Mori, a staff scientist in the LCLS hard X-ray group. “The same technology could be used not only for biological science but could also help data collection in other areas.”

    Micrograph of the microstructured chip, loaded with crystals for the investigation. Each square is a tiny crystal. (Philip Roedig/DESY)

    A Well-Rounded View of Life

    In the field known as structural biology, scientists examine the three-dimensional structure of biological molecules in order to work out how they function. This knowledge enhances our understanding of fundamental biological processes, such as the way substances are transported in and out of a cell, and can also inform drug development.

    “Knowing the three-dimensional structure of a molecule like a protein gives great insight into its biological behaviour,” explains co-author David Stuart, director of life sciences at the Diamond Light Source synchrotron facility in the United Kingdom and a professor at the University of Oxford. “One example is how understanding the structure of a protein that a virus uses to ‘hook’ onto a cell could mean that we’re able to design a defense for the cell to make the virus incapable of attacking it.”

    X-ray crystallography is by far the most prolific tool used by structural biologists and has already been used to determine the structure of thousands of biological molecules. Tiny crystals of the protein of interest are grown, and then illuminated using high-energy X-rays. The crystals diffract the X-rays in characteristic ways so that the resulting diffraction patterns can be used to deduce the spatial structure of the crystal – and hence of its components – on the atomic scale. However, protein crystals are nowhere near as stable and sturdy as salt crystals, for example. They are difficult to grow, often remaining tiny, and are easily damaged by the X-rays.

    “X-ray lasers have opened up a new path to protein crystallography, because their extremely intense pulses can be used to analyse even extremely tiny crystals that would not produce a sufficiently bright diffraction image using other X-ray sources,” says co-author Armin Wagner from Diamond Light Source. However, each of these microcrystals can only produce a single diffraction image before it evaporates as a result of the X-ray pulse. To perform the structural analysis, though, hundreds or even thousands of diffraction images are needed. In such experiments, scientists therefore inject a fine liquid jet of protein crystals through an X-ray laser beam that pulses in a rapid sequence of extremely short bursts. Each time an X-ray pulse happens to strike a microcrystal, a diffraction image is produced and recorded.

    This method is very successful and has already been used to determine the structure of more than 80 biomolecules, the researchers point out in their paper. However, most of the sample material is wasted. “The hit rate is typically less than 2 percent of pulses, so most of the precious microcrystals end up unused in the collection container,” says Meents, who is based at the Center for Free-Electron Laser Science (CFEL) in Hamburg, a cooperation of DESY, the University of Hamburg and the German Max Planck Society. The standard method therefore typically requires several hours of beam time and significant amounts of sample material.

    Protein Crystals on a Chip

    In order to use the limited beam time and the precious sample material more efficiently, the team developed a new method. The scientists use a micro-patterned chip containing thousands of tiny pores to hold the protein crystals. The X-ray laser then scans the chip line by line, and ideally this allows a diffraction image to be recorded for each pulse of the laser.

    The research team tested its method on two virus samples using SLAC’s LCLS X-ray laser, which produces 120 pulses per second. They loaded their sample holder with a small amount of microcrystals of the bovine enterovirus 2 (BEV2), a virus that causes miscarriages, stillbirths and infertility in cattle, and which is very difficult to crystallise.

    In this experiment, the scientists achieved a hit rate – where the X-ray laser successfully targeted the crystal – of up to 9 percent, five times the hit rate of the previous method. Within just 14 minutes they had collected enough data to determine the correct structure of the virus – which was already known from other experiments – down to a scale of 2.3 angstroms.

    “To the best of our knowledge, this is the first time the atomic structure of an intact virus particle has been determined using an X-ray laser,” Meents says. “Whereas earlier methods at other X-ray light sources required crystals with a total volume of 3.5 nanoliters, or billionths of a liter, we managed using crystals that were more than 10 times smaller, having a total volume of just 0.23 nanoliters.”

    This experiment was conducted at room temperature; while rapidly cooling the protein crystals would protect them to some extent from radiation damage, this is not generally feasible when working with extremely sensitive virus crystals. Crystals of isolated virus proteins can, however, be frozen and in a second test, the researchers studied a viral protein called polyhedrin that makes up a viral occlusion body — a container used by certain virus species to protect up to several thousand virus particles at a time against environmental influences so they can remain intact much longer.

    From Room Temperature to a Deep Chill

    For the second test, the scientist loaded their chip with polyhedrin crystals and examined them using the X-ray laser while keeping the chip at temperatures below minus 180 degrees Celsius. Here, the scientists achieved a hit rate of up to 90 percent. In just 10 minutes they recorded more than enough diffraction images to determine the protein structure to within 2.4 angstroms.

    “For the structure of polyhedrin, we only had to scan a single chip that was loaded with four micrograms of protein crystals; that is orders of magnitude less than the amount that would normally be needed,” explains Meents.

    “Our approach not only reduces the data collection time and the quantity of the sample needed, it also opens up the opportunity of analysing entire viruses using X-ray lasers,” Meents sums up. The scientists now want to increase the capacity of their chip by a factor of ten, from 22,500 to some 200,000 micropores, and further increase the scanning speed to up to one thousand samples per second. This would better exploit the potential of the European XFEL, which will be able to produce up to 27,000 X-ray laser pulses per second, as well as an upgraded LCLS that is scheduled to come on line in the early 2020s and produce up to a million pulses per second. Furthermore, the next generation of chips will expose only those micropores that are targeted for analysis, to prevent the remaining crystals from being damaged by scattered radiation from the X-ray laser.

    Diamond scientists have collaborated with the team at DESY, with much of the development and testing of the micro-patterned chip being on Diamond’s I02 and I24 beamlines. Researchers from the University of Oxford, the University of Eastern Finland, the Swiss Paul Scherrer Institute, Lawrence Berkeley National Laboratory and SLAC were also involved in the research. LCLS is a DOE Office of Science User Facility.

    See the full article here .

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    SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.

  • richardmitnick 8:52 pm on June 1, 2017 Permalink | Reply
    Tags: Assembled arenavirus glycoprotein, , Hemorrhagic fever viruses, , , , , Tripod Shape Key to Future Vaccine Design, Up to 90 percent fatal in pregnant women, X-ray crystallography   

    From SLAC: “SLAC X-Ray Beam Helps Uncover Blueprint for Lassa Virus Vaccine” 

    SLAC Lab

    June 1, 2017

    The molecular structure of a Lassa virus protein provides the blueprints for vaccine design. (Ollmann Saphire Lab/The Scripps Research Institute)

    Erica Ollmann Saphire, professor of Immunology and Microbial Science at The Scripps Research Institute, during a visit of the Kenema Government Hospital, Sierra Leone, to study Lassa virus. (Kathryn Hastie/The Scripps Research Institute)

    An antibody from a human survivor (turquoise) is shown inactivating a Lassa virus surface protein. (Ollmann Saphire Lab/The Scripps Research Institute)

    A decade-long search ends at the Stanford Synchrotron Radiation Lightsource, where researchers from The Scripps Research Institute emerge with a clear picture of how the deadly Lassa virus enters human cells.


    Before Ebola virus ever struck West Africa, locals were continually on the lookout for another deadly pathogen: Lassa virus. With thousands dying from Lassa every year – and the potential for the virus to cause even larger outbreaks – researchers are committed to designing a vaccine to stop it.

    Now a team of scientists from The Scripps Research Institute (TSRI) has solved the structure of the viral machinery that Lassa virus uses to enter human cells.

    X-ray beams from the Stanford Synchrotron Radiation Lightsource (SSRL) at the Department of Energy’s SLAC National Accelerator Laboratory gave the team the final piece in a puzzle they sought to solve for over 10 years.

    Their study, published today in Science, is the first to show a key piece of the viral structure, called the surface glycoprotein, for any member of the deadly arenavirus family, and the new structure provides a blueprint to design a Lassa virus vaccine.

    “This was a tenacious effort – over a decade – to conquer a global threat,” said Erica Ollmann Saphire, a professor of Immunology and Microbial Science from TSRI and senior author of the new study.

    X-ray data for this study was collected at SLAC and the DOE’s Argonne National Laboratory.

    For the SLAC experiments, the researchers used a station at SSRL, a DOE Office of Science User Facility that has a strong program in biological X-ray crystallography. In this method, scientists prompt biological molecules to align and form a crystal, which they then study with powerful X-rays. The way the X-rays scatter off the crystal reveals the structure of the molecules inside – in 3-D and with atomic detail.

    “I am proud of SSRL’s strong partnership with TSRI and our involvement in this project that utilized the bright X-ray microbeams and high level of automation at Beam Line 12-2 to obtain the necessary data,” said SSRL senior staff scientist Aina Cohen. “This structure provides key information towards engineering an effective vaccine against Lassa, enabling the infected to combat the immunosuppressive traits of this virus, which is estimated to kill tens of thousands of people each year.”

    It Started with a Thesis

    The effort began with TSRI staff scientist Kathryn Hastie, the lead author of the study. In 2007, then a grad student in Ollmann Saphire’s lab, she told her thesis committee she wanted to solve the structure of the assembled arenavirus glycoprotein, something never done before. She hoped to create a map of the target on the virus where antibodies need to attack – a key step in developing a vaccine.

    Such maps can be obtained with X-ray crystallography, but the method depends on having a stable protein. Yet, all the Lassa virus glycoprotein wanted to do was fall apart.

    The problem was that glycoproteins are made up of smaller subunits. Other viruses have bonds that hold the subunits together, “like a staple,” Hastie said. Arenaviruses don’t have that staple; instead, the subunits just floated away from each other whenever Hastie tried to work with them.

    Another challenge was to recreate part of the viral lifecycle in the lab – a stage when Lassa’s glycoprotein gets clipped into two subunits. “We had to figure out how to get the subunits to be sufficiently clipped, which is necessary to make the biologically functional assembly, and also where to put an engineered staple to make sure they stayed together,” Hastie said.

    Partnering with West Africa

    As Hastie tackled those challenges from her lab bench in San Diego, staff at the Kenema Government Hospital in Sierra Leone labored on the front lines of the ongoing fight against Lassa.

    Until the 2014–15 Ebola virus outbreak, Kenema was the only hospital in the world to have a special ward dedicated to treating hemorrhagic fever viruses. Staff at the clinic – from the nurses to the ambulance drivers – are all Lassa survivors, which gives them immunity to the disease. The TSRI scientists have a long-term collaboration with Kenema as part of a research program run by Tulane University that provided them with antibodies from survivors of Lassa fever. These antibodies could inactivate the virus, and they provided lifesaving protection to animal models. These were the kinds of antibodies researchers are hoping to elicit with a future Lassa virus vaccine.

    In 2009, Hastie got to visit Kenema on a trip with Ollmann Saphire.

    “I had been working on the project for two years with very little success at that point,” Hastie said. “Going to West Africa showed me how important it was to keep going.”

    Like Ebola virus, Lassa fever starts with flu-like symptoms and can lead to debilitating vomiting, neurological problems and even hemorrhaging from the eyes, gums and nose. The disease is 50 to 70 percent fatal—and up to 90 percent fatal in pregnant women.

    “Studying Lassa is critically important. Hundreds of thousands of people are infected with the virus every year, and it is the viral hemorrhagic fever that most frequently comes to the United States and Europe,” said Ollmann Saphire. “Kate’s study needed to be done.”

    Tripod Shape Key to Future Vaccine Design

    By creating mutant versions of important parts of the molecule, Hastie engineered a version of the Lassa virus surface glycoprotein that didn’t fall apart. She then used this model glycoprotein as a sort of magnet to find antibodies in patient samples that could bind with the glycoprotein to neutralize the virus.

    With this latest study she solved the structure of the Lassa virus glycoprotein, bound to a neutralizing antibody from a human survivor.

    Her structure showed that the glycoprotein has two parts. She compared the shape to an ice cream cone and a scoop of ice cream. A subunit called GP2 forms the cone, and the GP1 subunit sits on top. They work together when they encounter a host cell. GP1 binds to a host cell receptor, and GP2 starts the fusion process to enter that cell.

    The new structure also showed a long structure hanging off the side of GP1—like a drip of melting ice cream running down the cone. This “drip” holds the two subunits together in their pre-fusion state.

    Zooming in even closer, Hastie discovered that three of the GP1-GP2 pairs come together like a tripod. This arrangement appears to be unique to Lassa virus. Other viruses, such as influenza and HIV, also have three-part proteins (called trimers) at this site, but their subunits come together to form a pole, not a tripod. The structure is also important because it can be used as a model to conquer related viruses throughout the Americas, Europe and Africa for which no equivalent structure yet exists.

    “It was great to see exactly how Lassa was different from other viruses,” said Hastie. “It was a tremendous relief to finally have the structure.”

    This tripod arrangement offers a path for vaccine design. The scientists found that 90 percent of the effective antibodies in Lassa patients targeted the spot where the three GP subunits came together. These antibodies locked the subunits together, preventing the virus from gearing up to enter a host cell.

    A future vaccine would likely have the greatest chance of success if it could trigger the body to produce antibodies to target the same site.

    Ollmann Saphire explained that Hastie accomplished something unique in structural biology. “The research started from scratch with the native, wild-type viruses in patients in a remote clinic—and went all the way to developing a basis for vaccine design. And the work was done almost entirely by one woman.”

    Moving Forward with a Lassa Vaccine

    The next step is to test a vaccine that will prompt the immune system to target Lassa’s glycoprotein.

    As director of the Viral Hemorrhagic Fever Immunotherapeutic Consortium, Ollmann Saphire is already coordinating with her partners at Tulane and Kenema to bring a vaccine to patients.

    The Coalition for Epidemic Preparedness Innovations, an international collaboration that includes the Wellcome Trust and the World Health Organization as partners, has recently named a vaccine for Lassa virus as one of its three top priorities. “The community is keenly interested in making a Lassa vaccine, and we think we have the best template to do that,” said Ollmann Saphire.

    She added that with Hastie’s techniques for solving arenavirus structures, researchers can now get a closer look at other hemorrhagic fever viruses, which cause death, neurological diseases and even birth defects around the world.

    Ollmann Saphire added that beamlines such as 12-2 at SSRL, which provided the X-ray beam used to finally determine the Lassa virus glycoprotein structure, along with its recent detector upgrades, are essential for ongoing advances in structural biology.

    “This research highlights the power of crystallographic techniques that rely on advanced synchrotron facilities to combat the most challenging biological problems. The support of the DOE’s Office of Science Biological and Environmental Research, the National Institutes of Health and private institutions such as TSRI enables us to make these resources available to the wider biomedical community,” Cohen said.

    In addition to Ollmann Saphire and Hastie, the following authors contributed: Michelle A. Zandonatti of TSRI; James E. Robinson and Robert F. Garry of Tulane University; Lara M. Kleinfelter and Kartik Chandran of the Albert Einstein College of Medicine; and Megan L. Heinrich, Megan M. Rowland and Luis M. Branco of Zalgen Labs.

    The new study included (left to right) first author Kathryn M. Hastie, senior author Erica Ollmann Saphire and co-author Michelle A. Zandonatti of The Scripps Research Institute. (Photo by Madeline McCurry-Schmidt.)

    The study was supported by the National Institutes of Health and an Investigators in Pathogenesis of Infectious Diseases Award from the Burroughs Wellcome Fund. Research funding for the SSRL Structural Molecular Biology Program was provided by the DOE Office of Science and the National Institutes of Health, National Institute of General Medical Sciences.

    See the full article here .

    See the Scripps press release here .

    Please help promote STEM in your local schools.

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    SLAC Campus
    SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.

  • richardmitnick 9:23 am on December 15, 2016 Permalink | Reply
    Tags: , , , , , , X-ray crystallography,   

    From Stanford: “Masters of Crystallization” 

    Stanford University Name
    Stanford University

    March 24, 2016 [Stanford just put this in social media 12.14.16.]
    Glennda Chui

    When molecules won’t crystallize and technology confounds, who you gonna call?

    Macromolecular Structure Knowledge Center at Stanford’s Shriram Center. From left: Ted Li, T.J. Lane, MSKC Director Marc C. Deller, Nick Cox, Timothy Rhorer, Zachary Rosenthal.

    Researcher Ted Li examines a sample tray full of protein crystals under a microscope. Photo: SLAC National Accelerator Laboratory.

    Biology isn’t just for biologists anymore. That’s nowhere more apparent than in the newly furnished lab in room 097 of the Shriram Center basement, where flasks of bacterial and animal cells, snug in their incubators, are churning out proteins destined for jobs they may not have done in nature.

    Researchers who use this lab span a broad range of backgrounds and interests: Chemists searching for novel antibiotics. Chemical engineers developing biofuels. Doctors seeking new treatments for diabetes.

    Most of these highly skilled researchers have one thing in common: They have no idea how to grow the proteins and other large biomolecules that are essential to their research or how to prepare those proteins for X-ray studies that will reveal their structure and function.

    That’s where Marc Deller comes in.

    “I’m the lab manager, scientist, lab cleaner — I do everything, and I help people who don’t know how to use the equipment,” says Deller, who arrived in August to establish and direct the Macromolecular Structure Knowledge Center (MSKC). “I’m pretty much unboxing things every day and trying to get things plugged in.”

    With a doctorate from Oxford and years of protein-wrangling experience, he’s here to help Stanford faculty and students grow, purify and crystallize proteins and other big biomolecules so they can be probed with the SSRL synchrotron or the LCLS X-ray laser at SLAC National Accelerator Laboratory, just up the hill.

    SLAC SSRL Tunnel


    SLAC jointly funds the center with Stanford ChEM-H, an interdisciplinary institute aimed at understanding human biology at a chemical level, and the services offered at MSKC augment help available from the expert staff at the SLAC X-ray facilities.

    X-ray crystallography has been a revolutionary tool for understanding how living things work, revealing the structures of more than 100,000 proteins, nucleic acids and their complexes over the past few decades and fueling the development of numerous life-saving medications.

    But it’s not always easy, as chemistry graduate student Ted Li can attest. The protein he’s studying — a natural catalyst found in soil bacteria that scientists hope to turn into an antibiotic factory — “is very resistant to crystallization. It’s very floppy and doesn’t want to pack,” says Li, who works in the lab of Chaitan Khosla, professor of chemistry and of chemical engineering. “So I need to find a way to force them to do that. Most of the things I’m doing these days are completely new to me, and Marc is my main mentor. He’ll actually go with me to SLAC and guide me in how to collect my data.”

    In its first six months, MSKC has already helped scientists with two dozen research projects, and Deller is eager to round up more. “From my experience of doing this for 20 years,” he says, “making the protein is definitely a bottleneck.”

    See the full article here .

    Please help promote STEM in your local schools.
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    Stem Education Coalition

    Leland and Jane Stanford founded the University to “promote the public welfare by exercising an influence on behalf of humanity and civilization.” Stanford opened its doors in 1891, and more than a century later, it remains dedicated to finding solutions to the great challenges of the day and to preparing our students for leadership in today’s complex world. Stanford, is an American private research university located in Stanford, California on an 8,180-acre (3,310 ha) campus near Palo Alto. Since 1952, more than 54 Stanford faculty, staff, and alumni have won the Nobel Prize, including 19 current faculty members

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