X-ray pulses reveal structure of viral cocoon
X-ray pulses reveal structure of viral cocoon
February 13, 2017
February 13, 2017
Scientists analyze smallest ever protein crystals
Arizona State University’s Biodesign Center for Applied Structural Discovery (BCASD) and an international team of scientists have used high-intensity X-ray pulses to determine the structure of the crystalline protein envelope of an insect virus. Their analysis reveals the fine details of the building blocks that make up the viral cocoon down to a scale of 0.2 nanometers (millionths of a millimeter) – approaching atom-scale resolution.
The tiny viruses with their crystal casing are by far the smallest protein crystals ever analyzed using X-ray crystallography. This opens up new opportunities in the study of protein structures, as the team headed by DESY’s Leading Scientist Henry Chapman from the Center for Free-Electron Laser Science reports in the Proceedings of the National Academy of Sciences (PNAS).
“The granulovirus attacks certain insects and kills them. This initially leaves it stranded inside the decaying host, so it has to protect itself, perhaps for years, against adverse environmental conditions such as heat, ultraviolet radiation and drought, until it is once again ingested by an insect. To achieve this, the virus wraps itself in a cocoon made of protein crystals, which only dissolve again once it reaches an insect’s gut,” explains Cornelius Gati from DESY, the lead author of the paper.
The researchers examined the cocoon of the Cydia pomonella granulovirus (CpGV), which infects the caterpillars of the codling moth (Cydia pomonella) and is used in agriculture as a biological pesticide. The virus is harmless to humans.
Petra Fromme, study co-author and director of BCASD notes the rapid pace of advance in structural discovery, owing to the improved ability of researchers to produce high quality nanocrystals and more precisely analyze the resulting diffraction patterns produced when samples are subjected to the brilliant X-ray bursts delivered by X-ray Free Electron Laser (XFEL) instruments.
“So many important biological processes, like how we get sick or how plants capture sunlight, have been very difficult to study at the molecular level,” says Fromme. “This is a beautiful example of nature providing a clue to help scientist build better and smaller nanocrystals that will ultimately help us solve relevant and important problems in our world.”
In 2011, Fromme along with ASU professor of Physics, John Spence, and their collaborators, pioneered a method known as serial femtosecond crystallography. The technique is used to examine thousands of nanocrystals, using X-ray pulses at the femtosecond scale, (a millionth of a billionth of a second). Although the nanocrystals are vaporized by the X-ray pulse, the time frame is so short that a diffraction image is obtained before sample destruction occurs.
“With the success of this new study, we have made a major stride toward the goal of analyzing individual molecules,” says Spence, who also participated in the new work, along with fellow ASU researchers Nadia A. Zatsepin, Uwe Weierstall, R. Bruce Doak, Raimund Fromme, Ingo Grotjohann, Shibom Basu, Daniel James, Christopher Kupitz, Kimberly Rendek and Dingjie Wang.
Scientists are interested in the spatial structure of proteins and other biomolecules because this sheds light on the precise way in which they work. This has led to a specialized science known as structural biology. “Over the past 50 years, scientists have determined the structures of more than 100,000 proteins,” says Chapman, who is also a professor of physics at the University of Hamburg. “By far the most important tool for this is X-ray crystallography.”
One challenge, however, has been producing nanocrystals appropriate for this research. Many proteins do not readily align to form crystals, because that is not their natural state. The smaller the crystals that can be used for the analysis, the easier it is to grow them, but the harder it is to measure them. “We are hoping that in future we will be able to dispense altogether with growing crystals and study individual molecules directly using X-rays,” says Chapman, “so we would like to understand the limits.”
The virus particles used in the new study provided the smallest protein crystals ever used for X-ray structure analysis. The occlusion body (the virus “cocoon”) has a volume of around 0.01 cubic micrometers, about one hundred times smaller than the smallest artificially grown protein crystals that have until now been analyzed using crystallographic techniques.
To break this limit in crystal size, an extremely bright X-ray beam was needed, which was obtained using an XFEL, in which a beam of high-speed electrons is guided through a magnetic undulator causing them to emit laser-like X-ray pulses.
The scientists used the free-electron laser Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory in the U.S. and employed optics to focus each X-ray pulse to a similar size as one of the virus particles. Directing the entire power of the FEL onto one tiny virus exposed it to the tremendous radiation levels, equivalent to 1.3 billion Grays. (For comparison: the lethal dose for humans is around 50 Grays.)
The FEL dose was certainly lethal for the viruses too—each was completely vaporized by a single X-ray pulse. But the femtosecond-duration pulse carries the information of the pristine structure to the detector and the destruction of the virus occurs only after the passage of the pulse. The analysis of the recorded diffraction showed that even tiny protein crystals which are bombarded with extremely high radiation doses can still reveal their structure on an atomic scale.
“Simulations based on our measurements suggest that our method can probably be used to determine the structure of even smaller crystals consisting of only hundreds or thousands of molecules,” reports Chapman, who is also a member of the Hamburg Center for Ultrafast Imaging (CUI).
Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser; Cornelius Gati et al.; PNAS, 2017; DOI: 10.1073/pnas.1609243114
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About Arizona State University
Arizona State University is the largest public research university in the United States under a single administration, with total student enrollment of more than 70,000 in metropolitan Phoenix, the nation’s sixth-largest city. ASU is creating a new model for American higher education, an unprecedented combination of academic excellence, entrepreneurial energy and broad access. This New American University is a single, unified institution comprising four differentiated campuses positively impacting the economic, social, cultural and environmental health of the communities it serves. Its research is inspired by real-world application, blurring the boundaries that traditionally separate academic disciplines. ASU champions intellectual and cultural diversity and welcomes students from all 50 states and more than 120 nations. www.asu.edu.
About the Biodesign Institute at ASU
The Biodesign Institute at Arizona State University works to improve human health and quality of life through its translational research mission in health care, energy and the environment, global health and national security. Grounded on the premise that scientists can best solve complex problems by emulating nature, Biodesign serves as an innovation hub that fuses previously separate areas of knowledge to serve as a model for 21st century academic research. By fusing bioscience/biotechnology, nanoscale engineering and advanced computing, Biodesign’s research scientists and students take an entrepreneurial team approach to accelerating discoveries to market. They also educate future generations of scientists by providing hands-on laboratory research training in state-of-the-art facilities for ASU. biodesign.asu.edu
About the Biodesign Center for Applied Discovery
The Center for Applied Structural Discovery (CASD) unites a team of complementary research professionals from a wide range of disciplines, including biology, chemistry, physics and engineering, to develop and apply groundbreaking technologies and methodologies to accelerate the rate at which we discover the structure and associated function of biomolecules. Breakthrough knowledge will form the basis for fast progress on the path to technical innovations that improve human health and provide plentiful clean energy and food for future generations. The CASD mission is to develop new revolutionary techniques that reveal the structure and dynamics of biomolecules towards new visionary discoveries in medicine and energy conversion. biodesign.asu.edu/applied-structural-discovery
Deutsches Elektronen-Synchrotron (DESY) is the leading German accelerator centre and one of the leading in the world. DESY is a member of the Helmholtz Association and receives its funding from the German Federal Ministry of Education and Research (BMBF) (90 per cent) and the German federal states of Hamburg and Brandenburg (10 per cent). At its locations in Hamburg and Zeuthen near Berlin, DESY develops, builds and operates large particle accelerators, and uses them to investigate the structure of matter. DESY’s combination of photon science and particle physics is unique in Europe. To learn more, please visit http://www.desy.de/
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 U.S. Department of Energy Office of Science. SLAC National Accelerator Laboratory is supported by the Office of Science of the U.S. Department of Energy. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit: www.slac.stanford.edu