The ability to turn sunlight into energy is one of nature's most remarkable achievements. The scientists understand the basic process of photosynthesis, but many important details are not yet known.
That is changing now.
In a new study conducted by Petra Fromme and Nadia Zatsepin of the Biodesign Center for Applied Structural Discovery, the School of Molecular Sciences and the Department of Physics of the ASU, the researchers studied the structure of Photosystem I (PSI) with ultrashort X-ray pulses at the European Free-Electron X-Ray Laser (EuXFEL) in Hamburg.
PSI is a large biomolecular system that serves as a solar energy converter to convert solar energy into chemical energy. Photosynthesis provides energy for all complex life on Earth and provides the oxygen we breathe. Advances in deciphering the secrets of photosynthesis promise to improve agriculture and support the development of next-generation solar energy storage systems that combine the efficiency of nature with the stability of human-developed systems.
"This work is as important as it shows the first proof of concept of megahertz serial crystallography with one of the largest and most complex membrane proteins of photosynthesis: photosystem I," says Fromme. "The work paves the way for time-resolved studies at the EuXFEL to determine molecular films of the light-driven pathway of electrons in photosynthesis or to visualize how cancer drugs attack defective proteins."
The recently launched EuXFEL is the first to use a superconducting linear accelerator that offers exciting new features, including very fast megahertz repetition rates of its X-ray pulses – over 9000 times faster than any other XFEL – with less impulses are one millionth of a second apart. With these incredibly short x-rays, researchers will be able to absorb molecular films of basic biological processes much faster, and they will likely impact different areas, including medicine and pharmacology, chemistry, physics, materials science, energy research, environmental studies, electronics, nanotechnology and photonics. Petra Fromme and Nadia Zatsepin are corresponding authors of the paper published in the current issue of the journal Nature Communications .
Strength in Numbers
Fromme is the director of the Biodesigns Center for Applied Structural Discovery (CASD) and leads the team's experimental team efforts, while Zatsepin led the XFEL data analysis team.
"This is an important milestone in the development of femtosecond serial crystallography, based on the well-coordinated commitment of a large, multidisciplinary, international team and years of development across diverse fields," said Zatsepin, a former research associate at the ASU Institute for Physics and Biodesign CASD and now Senior Research Fellow at La Trobe University in Australia.
] Christopher Gisriel, co-author of the paper, worked as a postdoctoral fellow in the Religious Laboratory on the project and is thrilled with the project. "The rapid data collection in femtosecond serial crystallographic experiments makes this revolutionary technique accessible to those interested in the structure-function relationship of enzymes, as illustrated by our recent publication in Nature Communications which shows that even the most challenging and complex protein structures can be solved by serial femtosecond crystallography while data is collected at a megahertz repetition rate. "
" It's very exciting to see how the hard work of many people doing this Project has materialized, "says Jesse Coe, co-first author, who graduated with a Ph.D. in biochemistry of ASU. "This is a big step in the right direction to better understand the over billion-year-old process of electron transfer in nature."
An XFEL (for X-ray laser with free electrons) delivers X-ray light that is one billion times brighter than conventional X-ray sources. The brilliant, laser-like X-ray pulses are generated by electrons that are accelerated to nearly the speed of light and passed through the gap between a series of alternating magnets, a device known as an undulator. The undulator forces the electrons to wobble and join together to form individual packages. Each of the perfectly synchronized shaking electron packets emits a strong, short X-ray pulse on the electron flight path.
In femtosecond serial crystallography, a beam of protein crystals is injected into the pulsed XFEL beam at room temperature. provide structural information in the form of diffraction patterns. With these patterns, scientists can determine images of proteins at atomic scale under native conditions, paving the way for precise molecular films of molecules at work.
X-rays damage biomolecules, a problem that has plagued structural design efforts for decades, with the biomolecules needing to be frozen to limit the damage. However, the X-ray flashes generated by an XFEL are so short – they only last femtoseconds – that the X-ray scattering of a molecule can be recorded like a fast camera shutter before it is destroyed. As a reference point, a femtosecond is one millionth of a billionth of a second, the same ratio as a second to 32 million years.
Only five are currently available due to the sophistication, size, and cost of XFEL equipment. Such experiments are a serious bottleneck for researchers worldwide, as typically only one experiment per XFEL can be performed. Most XFELs generate X-ray pulses between 30 and 120 times per second, and it can take several hours to days to capture the data necessary to determine a single structure, let alone a series of images in a molecular film. The EuXFEL is the first to use a superconducting linear accelerator in its design that enables the fastest sequence of X-ray pulses from all XFELs, significantly reducing the time it takes to determine the individual structures or frames of the film.
High risk, high reward
Since the sample is extinguished by the intense X-ray pulses, it must be refilled in time for the next X-ray pulse so that PSI crystals must be released 9,000 times faster at the EuXFEL as with previous XFELs – with a jet speed of about 50 meters per second (160 feet per second), like a microfluidic fire hose. This has been a challenge because large amounts of the precious protein in uniform crystals are required to achieve these high jet velocities and to avoid blocking the sample delivery system. Large membrane proteins are so difficult to isolate, crystallize, and deliver to the jet that it was not known if this important class of proteins could be screened at the EuXFEL.
The team developed new methods that enabled a large PSI The complex consists of 36 proteins and 381 cofactors, which include 288 chlorophylls (which are the light-absorbing green pigments). It has more than 150,000 atoms and is more than 20 times larger than previous proteins studied on the EuXFEL to determine its structure at room temperature to a remarkable resolution of 2.9 angstroms – a significant milestone.
Billions of microcrystals of PSI membrane protein derived from cyanobacteria had to be cultured for the new study. Rapid crystal growth from nanocrystals was required to ensure substantial uniformity of crystal size and shape. PSI is a membrane protein, a class of proteins of great importance, whose characterization is known to be difficult. Their sophisticated structures are embedded in the lipid bilayer of the cell membrane. Typically, they must be carefully and fully actively isolated from their natural environment and converted to a crystalline state in which the molecules aggregate into crystals but retain their original function.
In the case of PSI, this is achieved by extraction with very mild detergents that replace the membrane and surround the protein like a pool inner tube that mimics the native membrane environment and keeps the PSI fully functional once packaged in the crystals , When the researchers illuminate the green pigments (chlorophylls) that are picked up by the antenna system of the PSI, the energy is used to shoot an electron through the membrane.
In order to keep the PSI fully functional, the crystals are only weakly packaged with 78% water, making them soft as a piece of butter in the sun and making it difficult to handle these fragile crystals. "To isolate, characterize and crystallize one gram of PSI or one billion billions of PSI molecules, because the experiments in their fully active form were a tremendous effort by the students and researchers in my team," says Fromme. Higher repetition rates and novel sample delivery systems lead to one drastic reduction of sample consumption.
Diffraction data recording and analysis was another challenge: A unique X-ray detector developed by EuXFEL and DESY to meet the requirements of structural biology studies at EuXFEL: the adaptive Gain Integrating Pixel Detector (AGIPD) AGIPD has a diameter of less than a hundredth of an inch and contains 352 analog memory cells that allow the AGIPD to capture megahertz rate data over a wide dynamic range, but to obtain accurate crystallographic data from microcrystals of large membrane proteins Compromise between spatial resolution and data sampling required.
The requirement for higher resolution data acquisition with the current detector size could prevent useful processing of the crystallographic data since the diffraction spots are insufficiently resolved by the pixels of the X-ray detector t, "warns Zatsepin". However, what the AGIPD is capable of in terms of data rates and dynamic range is unbelievable. "
The novel data reduction and crystallographic analysis software has been specially developed Since the first high-resolution XFEL experiment in 2011, it has come a long way to tackle the challenges that characterize the massive data sets in XFEL crystallography Development of employees led by CFEL, DESY and ASU.
"Our software and DESY's high-performance computing capabilities are being put to the test with the unprecedented amounts of data generated on the website EuXFEL. It is always exciting to push the boundaries of the state of the art, "adds Zatsepin.
Membrane Proteins: floppy but fearsome
Membrane proteins like PSI – named after their names Cell membranes – critical to all life processes such as respiration, nerve function, food intake, and cell-cell signaling, are also the most important pharmaceutical drug targets because they are located on the surface of each cell All common drugs target membrane proteins Developing more effective drugs with fewer side effects therefore requires understanding how certain drugs bind to their target proteins, how detailed their structural conformations and dynamic activities are.
Despite their enormous importance in biology, membrane protein structures make up less than 1% of all so far dissolved protein structures, since they b The most important advances in crystallographic methods, such as the advent of membrane protein megahertz femtosecond crystallography, will undoubtedly have a significant impact on science.
It takes a village
These Recent Achievements Without the tireless efforts of a dedicated team of nearly 80 researchers from 15 institutions, including ASU, European XFEL, DESY, Center for Ultrafast X-ray Research, Hauptman-Woodward Institute, SUNY Buffalo, SLAC, University of Hamburg, University of Göttingen, Hungarian Academy of Sciences, University of Tennessee, Lawrence Livermore National Laboratory, University of Southampton, Hamburg University of Technology, University of Wisconsin. The research group included US staff at Science and Technology Center NSF BioXFEL and a group of international staff, including Adrian P. Mancuso and Romain Letrun, chief scientists of beamline EuXFEL, and Oleksandr Yefanov and Anton Barty of CFEL / DESY, who worked closely together ASU team on the complex data analysis.
First published results of a new X-ray laser
Chris Gisriel et al., Membrane Protein Megahertz Crystallography at the European XFEL, Nature Communications (2019). DOI: 10.1038 / s41467-019-12955-3
Photosynthesis seen in a new light by rapid x-ray pulses (2019, 8 November)
retrieved on November 8, 2019
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