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Instrumentation Advances Expand the Reach of X-ray Free Electron Lasers

Monday, January 5th 2015

Femtosecond crystallography (FX) is especially suitable for studying radiation sensitive enzymes that require metals for their function, as the extremely short and bright X-ray pulses can produce a diffraction image before any atomic motions can occur in the crystal. This cutting edge method is capable of extending our capacity to study smaller, more fragile crystals and determine the catalytic structures of biologically relevant macromolecules.

In conventional X-ray crystallography experiments, one crystal is mounted on a goniometer, which is then used to rotate the sample in the X-ray beam. The short and bright X-ray pulses produced by the free-electron laser (XFEL) at the SLAC National Acceleratory Laboratory’s Linac Coherent Light Source (LCLS) damage or destroy crystals nearly instantaneously, requiring tens of thousands of crystals to be used in experiments. Recently, researchers in the Physical Biosciences Division (PBD) of the Lawrence Berkeley National Laboratory (Berkeley Lab) collaborated on a technique that will extend the ability of scientists to perform efficient and flexible FX experiments.

James Holton

James Holton

Currently, most FX researchers use injectors to deliver a continuous stream of crystals to the beam, which wastes a large portion of material. Alternate methods are being developed to expose a single drop of the crystalline material to the beam at one time, but these have not been perfected. In an article published on December 2, 2014 in PNAS, lead author Aina Cohen of SLAC describes an experiment using a goniometer-based method to mount samples in the path of the FEL. James Holton, PBD Biophysicist Faculty Scientist and the director of Beamline 8.3.1 at the Advanced Light Source (ALS), assisted in designing the experiment, which utilized equipment that was developed previously at SLAC’s LCLS and Stanford Synchrotron Radiation Light Source (SSRL). “Ever since the Braggs did their original X-ray crystallography work in 1914, crystals have been rotated during data collection to smooth over a myriad of difficulties. Now that XFEL pulses are far too fast to do this, Dr. Cohen and I had to return to these first principles in designing the data collection protocol,” Holton said. The resulting highly automated system uses specialized sample containers and customized software, allowing for efficient data collection and decreased crystalline sample consumption.

Nicholas Sauter, middle, pointing to a monitor during an experiment at SLAC. Photo by Fabricio Sousa/SLAC.

Nicholas Sauter, middle, pointing to a monitor during an experiment at SLAC. Photo by Fabricio Sousa/SLAC.

To compare the utility of this setup, researchers collected data from two types of protein crystals (two macromolecular complexes and two metalloenzymes) using both goniometer- and injector-based delivery. Nicholas Sauter, Computational Staff Scientist in PBD, along with two postdoctoral researchers in his group, Aaron Brewster and Johan Hattne (now a Research Specialist at Janelia Farm Research Campus), were part of the team that analyzed these data. “Most of the FX experiments were done with a moving stream of crystals, but that made it hard to collect and process data,” said Sauter. “Goniometer methods are familiar to all crystallographers. The fact that they work with femtosecond X-ray pulses makes this technique accessible to everyone’s protein crystals.” cctbx.xfel, open-source software for free-electron laser data processing previously developed by Sauter and his group, was used during the experiment for quasi-real time data analysis.

Aside from SLAC and Berkeley Lab, Cohen’s research team was made up of participants from the Stanford University School of Medicine, the University of Pittsburgh School of Medicine, Howard Hughes Medical Institute, Montana State University, and the University of California, San Francisco. Employing automated goniometer-based instrumentation allowed researchers to determine high-resolution structures of four molecules using FX on just a fraction of the crystals typically required using injector-based methods. “In doing this, we sparked a revolution in how we think about X-ray diffraction data all around the world, and processing software has been improving by leaps and bounds ever since,” said Holton. “The original protocol we developed is already obsolete, and soon even synchrotron-based data collection may follow suit, taking advantage of the fundamentally superior data quality from non-rotating crystals for the first time in 100 years.” By reducing both crystalline material sample and data processing requirements, the team has effectively decreased the overall cost and time needed to perform these cutting-edge scientific experiments and has taken steps that will eventually lead to wider use of this technology.

Microbes-to-Biomes (M2B) Initiative Launches

Monday, December 15th 2014

The Labwide initiative, Microbes-to-Biomes (M2B), has kicked off with five projects funded through the Laboratory Directed Research and Development (LDRD) program, and a new website to chronicle news and advancements in M2B’s research mission. The M2B initiative is designed to explore and reveal the interactions of microbes with one another and with their environment – interactions that are vital to the Earth’s future. To jumpstart the discovery process, M2B is targeting two key systems: the soil-plant biome and the gut microbiome. Research areas include Food and Fuel Production, Carbon Management, Environmental Stewardship, and Health and Environment.

Two PBD scientists, Adam Deutschbauer and Trent Northen, are co-PIs on separate projects within the Soil-Plant Biome portion of M2B. Deutschbauer will be partnering with Matthew Blow (Genomics Division & Joint Genome Institute) to engineer phosphate solubilizing planAdam Deutschbauert-associated bacteria. This would allow the bacteria that are already co-localized with plants to convert existing phosphorus sources in the soil to soluble forms, thereby making it available for plant uptake. Increasing the amount of soluble phosphorus in the soil would mean decreasing the use of costly and environmentally damaging phosphate fertilizers in high yield agriculture. Northen and Javier Ceja Navarro (Earth Sciences Division) will be leading a project looking at uncovering the Soil Metazoan Microbiome, a key compartment that is important for plant health and root carbon fixation. Metazoans are multicellular organisms of the Kingdom Animalia (also called Metazoa); 050814_Trent_NorthenHeadshot2-150x150two sub-groups of this Kingdom, arthropods (animals with an exoskeleton and segmented body) and nematodes (worms), function as ecosystem engineers. Through both physical and chemical transformation of soil they provide modified habitats for soil microorganisms, thereby playing an important role in the cycling of nutrients. Northen and Navarro will study metazoan-associated microorganisms with a specific focus on plant associated arthropods and nematodes with an eye to characterizing of the contribution of soil metazoans, and their microbiomes to nutrient cycling, and gaining a better understanding of the regulation of microbial activity by their environment, i.e. the metazoan host.

Steve SingerSteve Singer, a scientist in the Earth Sciences Division and Director of Microbial Communities at the Joint BioEnergy Institute, will be working with Tanja Woyke and Natalia Ivanova, both of the Genomics Division and Joint Genome Institute, to use function-driven genomics to capture carbon degrading microbes with fluorescent substrate bait. Together, they will apply novel approaches to experimentally capture bacteria that degrade plant-derived biopolymers (e.g., cellulose) for sequence-based characterization. Singer and his colleagues intend to greatly advance our understanding of soil carbon cycling, which will have implications on below-ground carbon storage and atmospheric C flux. This same information and microbial functional potential can also be harnessed to improve the breakdown of plants for the production of biofuels.


Looking to the Future Earns a Place in History

Friday, December 5th 2014

When presenting a new idea, formulating an experiment, or communicating research, all researchers build on the body of previously published literature. By citing earlier articles, authors lay the groundwork for their hypotheses, justify their results, and relay their methods using a common language. Therefore, the number of times articles are referenced by later publications indicates the relevance or importance for subsequent work.

Nature recently released a list of the 100 most-cited articles published between 1900 and 2014 as recorded in the Science Citation Index (SCI). It is not surprising that many of theNature Logo highest ranked papers describe methods or software programs essential to researchers. Of the almost 57 million items recorded by the SCI, nearly half have never been cited, more than 18 million have fewer than 10 citations, and approximately 13 million fall short of the 100-citation mark. To make it into the top 10, the articles need to have at least 12,119 citations, with the most cited paper of all having more than 305,000.

Paul Adams, Physical Biosciences Division Deputy for Science, was a lead co-author on one of the top 100 articles in 1998, when he was a postdoctoral scholar working with Axel CNSsolve logoBrunger at Yale University. The 69th -ranked paper describes Crystallography & NMR System (CNS), a software suite for automating macromolecular structure determination by either X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. “Unlike other programs at the time,” says Adams, “CNS provided the algorithms most commonly used at the time in structure determination in a flexible system.” This design and construction made CNS widely accessible to structural biologists and adaptable for use with other biophysical measurements, such as electron microscopy, neutron diffraction, and fiber diffraction.

After starting his own group at Berkeley Lab in 1999, Paul Adams formed an international collaboration that develops PHENIX (Python-based Heirarchical ENvironment for Integrated Xtallography), a next generation software package for automated macromolecular structure determination. “We have integrated componentphenix-logos for each of the major steps involved in solving, refining, and validating structures using X-ray crystallographic data,” says Adams. A modular architecture allows a team of 20 scientists to rapidly implement new features and develop program functions. “It is now possible to solve more than 50% of structures in an automated fashion,” Adams continues. “This means that more macromolecular structures are determined in a OLYMPUS DIGITAL CAMERAshorter amount of time so that researchers are free to solve increasingly difficult structures and maximize our collective understanding of macromolecular function.”

Since its introduction in 2000, the two primary articles describing PHENIX have together amassed over 5400 citations, more than Watson & Crick’s seminal paper describing the structure of DNA, and more than 1000 citations per year since 2012. The ease of use, improvement in structure quality, and flexible architecture leads to popularity among researchers. By improving the structure determination process, both of these programs facilitate exciting discoveries, ensuring many more future citations.

Berkeley Center for Structural Biology to Receive $5M from HHMI to Build a New Microfocus Crystallography Beamline

Friday, October 24th 2014

The Berkeley Center for Structural Biology (BCSB) has operated five beamlines at the Advanced Light Source (ALS) for more than ten years, helping hundreds of crystallographers to determine the structures of more than 1,000 proteins. Two of the BCSB’s beamlines (8.2.1 and 8.2.2) are funded by the Howard Hughes Medical Institute (HHMI) to support the cutting edge research of structural biologists, including those in the HHMI research community. To ensure that these crystallographers have access to state-of-the-art instrumentation and user support, HHMI has pledged $5 million to the BCSB over the next 3 years to construct a new cutting-edge microfocus macromolecular crystallography beamline.


Paul Adams and Corie Ralston, who worked together to bring in the funding for the GEMINI project, in front of the current HHMI beamlines at the ALS (top). Simon Morton, the scientist who came up with the original idea for GEMINI, prepares to align an x-ray beam (bottom photo).

Working closely with the ALS, the BCSB has developed a plan for GEMINI, a vanguard beamline that provides the structural biology capabilities required by researchers in the coming decade. “GEMINI will have cutting-edge X-ray technology to increase flux and provide a smaller beam focus, as well as advanced automation for sample handling and high performance detectors for data collection,” said Corie Ralston, Head of the BCSB. “HHMI believes, as we do, that this system will be synergistic, exceeding the capability of any currently available structural biology beamline. This kind of project also highlights the interdisciplinary collaborations made possible by the environment at Berkeley Lab. We have access to an extremely talented pool of people with different backgrounds and areas of expertise: Peter Zwart and Simon Morton (Physical Biosciences), Chuck Swenson (Engineering), and Christoph Steier (Accelerator and Fusion Research), to name just a few, who have formed a first-class team working together toward a common goal.”

Hundreds of users access the HHMI beamlines every year, solving high impact challenging problems. One of these discoveries has been made recently by HHMI investigator Jennifer Doudna, Faculty Biochemist in the Physical Biosciences Division, and Professor of Molecular and Cell Biology at UC Berkeley. Doudna used the BCSB beamlines, as well as ALS beamline 8.3.1, to solve the structure of Cas9, lending insight into the CRISPR/Cas9 system. As a result of this increased structural understanding, this bacterial genetic editing system is revolutionizing the field of molecular biology and gaining wide use in a variety of fields.

Some of the most challenging projects in structural biology today involve the study of membrane proteins, receptors, and large protein complexes. While characterizing these systems at a structural level is crucial to understanding their function, these proteins typically yield very small, weakly diffracting crystals. “Progress is being made to study these crystals using free electron lasers, but these are early days for this technology and the experiments are still difficult to perform,” said Paul Adams, Deputy Director of the Physical Biosciences Division and Division Deputy for Biosciences at the Advanced Light Source. Adams is an x-ray crystallographer who has been involved in free electron laser experiments, and he spoke of the opportunity GEMINI provides. “GEMINI helps address the need for solving structures from these challenging systems without resorting to an FEL.”

This transformation of the crystallography experiment is made possible by three major factors. First, development of technology for synchrotron beamlines has resulted in devices that deliver high power “off the shelf” components.

GEMINI brings together the latest technologies in x-ray optics, detectors, and precision sample handlers in one facility.

GEMINI brings together the latest technologies in x-ray optics, detectors, and precision sample handlers in one facility.

The combination of these new technologies with the experience and expertise of the ALS staff means that on-line real-time beam focusing can be realized, so that the spot size and shape can be focused to the exact size of each crystal. Second, GEMINI leverages two robotic sample mounters in a coordinated system that will bring sample exchange times down to seconds, making it home to the world’s fastest sample mounter. Third, technological advances in x-ray detectors now make it possible to collect data sets in minutes or seconds, with better signal-to-noise than the best CCD detectors currently available. Together, these advances will enable centering, rastering, and data collection from every sample within seconds or minutes. With GEMINI, sample exchange and data collection will be so fast that screening and data collection converge. Every crystal – no matter how small or poorly diffracting – can be evaluated and data collected.

The total cost for GEMINI is $10M over a period of three years. Already, the BCSB has secured a commitment of additional funding from industry partners, and several other organizations have expressed their interest. In addition, Berkeley Lab management recognizes the importance of this project to the mission of the Lab, and has committed resources for the infrastructure changes in the ALS ring required to accommodate GEMINI.

GEMINI evolved from the BCSB’s extensive experience with x-ray optics, automation and outstanding operational support. HHMI’s support for this project will help BCSB advance its vision, expertise and service and aid in providing the advanced technologies needed by structural biologists now and in the coming years.

Stimulating Insulin Production in the Fight Against Type-II Diabetes

Tuesday, September 23rd 2014

Adult-onset diabetes, characterized by abnormally high blood sugar, affects hundreds of millions of people worldwide. New treatments for this disease have centered on targeting the human receptor protein GPR40, since it can enhance sugar-dependent insulin secretion. TAK-875 is a drug developed by the company Takeda to stimulate insulin secretion by binding to this receptor. However, the structural information needed to fully understand this class of drugs has not been available until now.

In the September 4th issue of Nature, Takeda researchers reported the first three-dimensional structure of this important receptor-drug interaction. The structure was solved at the Advanced Light Source in the Berkeley Center for Structural Biology (Beamline 5.0.3) and reveals details about the mechanism of action of this drug. The results of this study point the way to better and more effective medications for diabetes.