Phycobilisomes attach to cellular membranes where the light-dependent reactions of photosynthesis take place. Changing the antenna complexes of the phycobilisomes can have dramatic and far-reaching consequences in cyanobacteria. Artificially modifying phycobilisomes by deleting certain genes in the cells caused structural defects in the cellular membranes and other cell physiology, allowing scientists to observe the resulting structural changes.
Starving the cyanobacteria for nitrogen naturally modifies the antenna complexes, causing the antenna to decrease in size and leading to significant cellular membrane modifications, because the cells break down the phycobilisomes and use them as an alternative nitrogen source to survive. By determining the extent of these changes, the team hopes to better understand the structure-function relationship between cellular organization and natural modification. These processes can be immediately reversed by restoring nitrogen to the cells. The researchers plan to compare these results to those recorded from their genetic studies to explore the differences between artificial and natural modifications and their effects on the intracellular makeup of cyanobacteria.
Instruments and Facilities Used: Photosynthetic Antenna Research Center (PARC), a DOE-funded Energy Frontier Research Center based at Washington University. Small angle neutron scattering (SANS) Bio-SANS instrument, beamline CG‑3, at Oak Ridge National Laboratory’s High Flux Isotope Reactor.
Oliver, R. C., et al. “Designing Mixed Detergent Micelles for Uniform Neutron Contrast.” The Journal of Physical Chemistry Letters 8(20), 5041–5046 (2017). [DOI:10.1021/acs.jpclett.7b02149].
Instruments and Facilities Used: Small angle neutron scattering (SANS): Bio-SANS beamline (CG3) of the High-Flux Isotope Reactor at Oak Ridge National Laboratory (ORNL). Recorded scattering data using MantidPlot software. Neutron contrast studies: ModULes for the Analysis of Contrast (MULCh) Variation Data at University of Sydney (smb-research.smb.usyd.edu.au/NCVWeb/).
O’Neill, H. M., et al. “Dynamics of Water Bound to Crystalline Cellulose.” Sci. Rep. 7, Article 11840 (2017). [DOI:10.1038/s41598-017-12035-w].
Instruments and Facilities Used: Deuterium labeling, neutron scattering, and molecular dynamics simulation; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer, at Oak Ridge National Laboratory (ORNL) Spallation Neutron Source (SNS). Structural characterization using small-angle neutron scattering (SANS) CG-3 Bio-SANS instrument at High Flux Isotope Reactor (HFIR) facility of ORNL and X-ray diffraction (XRD) analysis combined with SANS. Wide-Angle X-ray Diffraction (WAXD) using theta-theta goniometer Bruker D5005 instrument; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer at ORNL SNS; Molecular Dynamics (MD) Simulations (GROMACS software and the CHARMM C36 carbohydrate force field); and TIP4P water model.
Dhiman, I., et al. “Quantifying Root Water Extraction After Drought Recovery Using sub-mm In Situ Empirical Data.” Plant Soil 417, 1–17 (2017). [DOI:10.1007/s11104-017-3408-5].
Instruments and Facilities Used: Sequential neutron radiography using CG1-D beam line using cold neutrons, at High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. Neutron attenuation by plant samples was detected with a 25-μm lithium fluoride / zinc sulfide (LiF/ ZnS) scintillator linked to a charge coupled detector (CCD) camera system (iKon – L 936, Andor Technology plc., Belfast, U.K.). Roots scanned and dimensions measured using WinRhizo software (Regent Instruments Inc., Quebec, Canada.
Enough sunlight hits the Earth to power the planet many times over—if energy capture were more efficient. Thus, scientists have looked at nature’s means to understand photosynthesis in a sun-loving, soil-dwelling bacterium. A team of scientists has gained a fundamental understanding of the inner workings of this vital process and how it differs among plant systems. Their discovery may provide a new template for beginning organic-based solar panel design (i.e., “artificial leaves”) or possibly renewable biofuel applications.
But the team’s structural biologists are capturing freeze-frame images of crystallized proteins throughout the whole process of the simplest form of photosynthesis, in the single-celled heliobacteria, which are fundamentally different than plants. Instead of using water like plants, for example, heliobacteria use hydrogen sulfide and grow without oxygen; after photosynthesis, they give off the foul-smelling sulfur gas in place of oxygen. The scientists grew the “perfect” X-ray diffracting crystal charge after many initial trials. In this breakthrough study, they found an almost perfect symmetry in the heliobacter RC, which helps it gather every available photon of near-infrared light to build chlorophylls.
Gisriel, C., et al. “Structure of a symmetric photosynthetic reaction center–photosystem,” Science 357(6355), 1021–1025 (2017). [DOI:10.1126/science.aan5611].
Instruments and Facilities Used: X-ray macromolecular crystallograpy at Biodesign Center for Applied Structural Discovery at Arizona State University. Advanced Light Source at Berkeley Center for Structural Biology at Lawrence Berkeley National Laboratory. Structural Biology Center–CAT beamline of the Advanced Photon Source at Argonne National Laboratory.
Researchers successfully capped 7 years of work by obtaining a high-resolution (2.2 Å) structure of the membrane protein at the heart of the photosynthetic RC of H. modesticaldum. The structure revealed details such as the interactions between light-harvesting “antenna” molecules and the electron-transfer chain, where the initial steps required to convert photon energy into chemical energy occurs. With this data, the research team was able to make comparisons to other types of RCs, gaining new perspectives on the early evolution of photosynthesis and how nature optimized light-driven energy collection. This work will help unlock the secrets of photosynthesis, possibly leading to the development of cleaner, solar-based renewable energy.
Gisriel, C., et al. “Structure of a Symmetric Photosynthetic Reaction Center–Photosystem.” Science 357(6355), 1021–1025 (2017). [DOI:10.1126/science.aan5611].
Instruments and Facilities: Beamline 8.2.1 at Advanced Light Source at Lawrence Berkeley National Laboratory; Advanced Photon Source at Argonne National Laboratory.
Researchers relied on an X-ray-based technique known as “footprinting” to pinpoint the chemical connections between the bacterial protein and nanoparticles composed of iron and oxygen. The study identified a previously mapped, surprisingly small and weak binding site, but unknown was how the site bound to the metal-containing mineral because conventional techniques can’t see this binding process. Footprinting reveals interactions of proteins in a near-native environment. The protein selected for the study is from the metal-reducing bacterium Shewanella oneidensis, which “eats sugar and basically breathes minerals” when oxygen is unavailable. This study already is providing ideas on how to redesign these proteins to make better electronic connections and thus more sensitive bioelectronic sensors.
Fukushima, T., et al. “The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface.” J. Am. Chem. Soc. 139(36), 12647–12654, (2017). [DOI:10.1021/jacs.7b06560].
Instruments and Facilities Used: X-ray beamline 5.3.1 of the Advanced Light Source (ALS) to perform “footprinting” and mass spectrometry at the Biological Nanostructures Facility at the Molecular Foundry, both at Lawrence Berkeley National Laboratory (LBNL), and the LBNL-led DOE Joint BioEnergy Institute.
The work builds on the 2005 discovery that the bacteria produce toxic proteins, which they can transfer to their neighbors through direct contact to either kill or control them, possibly to gain better access to nutrients in densely populated microbial communities through a process called contact-dependent growth inhibition (CDI). Learning how the bacteria interact and communicate is helping to resolve the possibly different activities of the toxins, which “may affect different bacteria differently.” Found in soil and gut bacteria, as well as in human pathogens, some of these toxins of CDI systems are present, for example, in Pseudomonas aeruginosa, which is involved in lung disease.
The team obtained the molecular structures of proteins that belong to a three-part system of the NC101 strain of E. coli, which consists of the CDI toxin, its immunity protein, and its elongation factor (EF). The latter, known as EF-Tu, is a protein that plays a key role in protein synthesis. Knowing the protein structures of all three parts helps scientists understand their function. Discovery of the immunity protein has led scientists to suspect that the system’s purpose includes competition and signaling (intracommunication), as well as killing and controlling other bacteria. Actually, only a few molecules of the toxin “get into the neighboring cell,” so perhaps the toxin is “not meant to kill, but rather to control and communicate.” It can act on the transfer ribonucleic acid (tRNA) only under highly specific circumstances, and it is the first case seen where the EF is “needed for the toxin to function.” Researchers used high-bright X-rays to characterize, or identify, biological proteins and inspect chemical processes at the nanoscale level (i.e., one billionth of a meter).
Michalska, K., et al. “Structure of a Novel Antibacterial Toxin That Exploits Elongation Factor Tu to Cleave Specific Transfer RNAs.” Nucleic Acids Res. 45(17), 10306–10320 (2017). [DOI:10.1093/nar/gkx700].
Instruments and Facilities Used: Advanced Photon Source 19-ID beamline and Advanced Protein Characterization Facility at Argonne National Laboratory.
Mus, F., et al. “Structural Basis for the Mechanism of ATP-Dependent Acetone Carboxylation.” Nat. Sci. Rep. 7, Article 7234 (2017). [DOI:10.1038/s41598-017-06973-8].
Instruments and Facilities Used: X-ray crystallographic data from the Stanford Synchrotron Lightsource (SSLS) at SLAC National Accelerator Laboratory (SLAC). Structural data from macromolecular crystallography were measured on SSLS beamline 12-2; Advanced Photon Source (APS) at Argonne National Laboratory (ANL); and the Diamond Light Source, Oxfordshire, United Kingdom.
Bacterial DNA is characterized by regions of clustered regularly interspaced short palindromic repeats (CRISPRs) and associated Cas proteins (CRISPR-associated endonucleases). The CRISPR-Cas system has revolutionized gene editing by vastly simplifying the insertion of short snippets of new (“donor”) DNA into very specific locations of target DNA. Researchers in this study have discovered how Cas proteins recognize their target locations with such great specificity. They used x-ray crystallography to solve the structures of Cas1 and Cas2—responsible for DNA-snippet capture and integration—as the proteins were bound to synthesized DNA strands designed to mimic different stages of the process. The research also demonstrated how the system works in its native context as part of a bacterial immune system and how Cas proteins act as general-purpose molecular recording devices—tools for encoding information in genomes.
Cas1 appears to have evolved from a more “promiscuous” (less selective) type of enzyme that catalyzes the movement of DNA sequences from one position to another (a transposase). At some point, Cas1 acquired an unusual degree of specificity for a particular location in the bacterial genome, the CRISPR array. This specificity is critical to the bacteria, both for acquiring immunity and for avoiding genome damage caused by the insertion of viral fragments at the wrong location. The researchers wanted to learn how Cas1-Cas2 proteins recognize the target sequence to enable comparison with previously studied transposases and integrases (i.e., enzymes that catalyze the integration of donor DNA into target DNA) and to determine whether the proteins can be altered to recognize new sequences for custom applications.
The researchers crystallized Cas1-Cas2 in complex with preformed DNA strands that mimicked reaction intermediates and products. X-ray crystallography revealed that the structures showed substantial distortions in the target DNA, but there were surprisingly few sequence-specific contacts with the Cas1-Cas2 complex, and the DNA’s resulting flexibility produced disorder in the crystals. Attempts to model the DNA across the disordered sections showed that the DNA had to be even more distorted. Cryoelectron microscopy experiments, coupled with the crystallography data, confirmed that an accessor protein called the integration host factor (IHF) introduces an additional sharp bend in the DNA, bringing an upstream recognition sequence into contact with Cas1 to increase both the specificity and efficiency of integration. The architecture of the CRISPR integration complex suggests that subtle adjustment of the distance between Cas1 active sites could reprogram the system to recognize different target sites. Changes in its architecture could be exploited, thereby, for genome tagging applications and also may explain the natural divergence of CRISPR arrays in bacteria.
Wright, A. V., et al. “Structures of the CRISPR Genome Integration Complex,” Science 357(6356), 1113–1118 (2017). [DOI:10.1126/science.aao0679].
Instruments and Facilities Used: X-ray macromolecular crystallography; beamline 8.3.1; protein crystallography (PX); and scattering/diffraction at the Advanced Light Source at Lawrence Berkeley National Laboratory; Stanford Synchrotron Radiation Light Source 9-2 beamline.
Goodell, B., et al. “Modification of the Nanostructure of Lignocellulose Cell Walls via a Non-Enzymatic Lignocellulose Deconstruction System in Brown Rot Wood-Decay Fungi.” Biotechnol. Biofuels 10(1), 179 (2017). [DOI:10.1186/s13068-017-0865-2].
Instruments and Facilities Used: Small angle neutron scattering (SANS) – Bio-SANS at High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory (ORNL). Sum frequency generation (SFG) spectroscopy, broadband SFG system. Chelator-mediated Fenton treatments (CMF) and cellulase treatment. X-ray diffraction analysis (XRD) – PANalytical Empyrean diffractometer, The Netherlands, equipped with a Cu X-ray source. Attenuated total reflectance Fourier transform infrared analysis (ATR-FTIR): Nicolet 8700 FTIR Spectrometer (Thermo Scientific) equipped with a smart iTR diamond ATR unit, a KBr beam splitter, and a deuterated triglycine sulfate (DTGS) detector. Atomic force microscopy (AFM) of brown-rotted wood surfaces. Nanoscope IIIa AFM-Digital Instruments, Santa Barbara, California with three 5 µm scans. Transmission electron microscopy (TEM) – Philips CM12 TEM instrument (Philips, Eindhoven, The Netherlands); images recorded on Kodak 4489 negative film and the films subsequently scanned using an Epson Perfection Pro 750 film scanner.
One copy was determined to be functionally equivalent to the well-characterized OCP of Synechocystis cyanobacteria, dubbed OCP1. But the second, OCP2, was distinct in several key aspects. The researchers hypothesize that OCP2 and another bioinformatically identified protein, OCPx, reflect intermediate stages in the evolution of photoprotection in cyanobacteria.
Bao, H., et al. “Additional Families of Orange Carotenoid Proteins in the Photoprotective System of Cyanobacteria.” Nature Plants 3, Article 17089 (2017). [DOI:10.1038/nplants.2017.89].
Instruments and Facilities: X-ray macromolecular crystallography and diffraction at Advanced Light Source at Lawrence Berkeley National Laboratory.
With no approved vaccine and limited treatment for RSV, doctors prescribe the antiviral drug ribavirin only in the most severe cases because it is expensive and not very effective. Thus, most people with RSV receive only supportive care to make them more comfortable while their bodies fight off the virus. People with weakened immune systems face a tough fight with RSV. The researchers say that solving the structure of this elusive protein will enable them to see what the protein looks like and help them define what it does and how it does it. Ultimately, this capability and finding could lead to new targets for vaccine or drug development.
Scientists have long known that a nonstructural RSV protein (known as NS1) is key to the virus’s ability to evade the body’s immune response. However, its structure was unknown, so scientists were unable to determine exactly how the enigmatic NS1 interfered with the immune system. Using X-ray crystallography, the scientists determined the three-dimensional (3D) structure of NS1, and, in a detailed analysis of the structure, they identified a piece of the protein known as the alpha 3 helix, possibly critical for suppressing the immune response.
The researchers created different versions of this NS1 protein, with some having an intact and some with a mutated alpha 3 helix region. They tested the functional impact of helix 3 and created a set of viruses containing either the original or the mutant NS1 genes, measuring the effect on the immune response when they infected cells with these viruses. They found that viruses with the mutated helix region did not suppress the immune response, while the ones with the intact helix region did, globally modulating the immune response.
The findings also showed that the protein’s alpha 3 helix region was necessary for the virus to dial down the body’s immune response, giving the virus a better chance of surviving and multiplying—in other words, causing disease. Thus, a vaccine or treatment that targets the alpha 3 helix may supply what is needed to prevent immune suppression.
Chatterjee, S., et al. “Structural Basis for Human Respiratory Syncytial Virus NS1-Mediated Modulation of Host Responses.” Nat. Microbiol. 2, Article 17101 (2017). [DOI:10.1038/nmicrobiol.2017.101].
Instruments and Facilities Used: X-ray crystallography at the Advanced Photon Source at Argonne National Laboratory.
Plants are the chemists of the living world, producing hundreds of thousands of small molecules that provide protection—to screen sunrays, to poison plant eaters, to scent the air, to color flowers, and for much other vegetative business.
Called “secondary metabolites,” these chemicals are distinguished from “primary metabolites,” which are the essential building blocks of proteins, fats, sugars, and DNA. Secondary metabolites just smooth the way in life, but failure to make primary metabolites correctly and efficiently is fatal. Genes for enzymes in the molecular assembly lines of primary metabolism have duplicates, allowing more tolerance of mutations that might have destabilized the primary pathways because the originals were still on the job. With evolutionary constraints thus relaxed, synthetic machinery was able to accumulate enough mutations to do new chemistry.
Widely conserved, primary metabolism it was thought to remain unchanged across many different groups of organisms because it operates correctly and efficiently and because its products are necessary for life. But now, a collaborative team of scientists has caught primary metabolism in the act of evolving. In a comprehensive study of a primary-metabolism assembly line in plants, they discovered a key enzyme evolving from a canonical form possessed by most plants, through noncanonical forms in tomatoes, to a switch-hitting form found in peanuts, and finally committing to the novel form in some strains of soybeans. This feat is comparable to pulling the tablecloth out from under the dishes without breaking any of them. A collaborative study of this biochemical pathway resulted in the crystallization of the soybean enzyme to reveal how nature changed the way the protein works, also capturing plants “building a pathway that links the primary to the secondary metabolism,” to reveal evolutionary machinery that creates new molecules.
A new pathway discovered for making tyrosine is much less constrained than the old one, raising the possibility that carbon flow could be directed away from lignin to increase the yields of drugs or nutrients to levels that would allow them to be produced in commercial quantities. Though the scientists have found two different assembly lines for tyrosine, they have not determined why except in general terms. This work is important because it demonstrates that primary metabolism does evolve.
Schenck, C. A., et al. “Molecular Basis of the Evolution of Alternative Tyrosine Biosynthetic Routes in Plants.” Nat. Chem. Biol. 13, 1029–1035 (2017). [DOI:10.1038/nchembio.2414].
Instruments and Facilities Used: X-ray macromolecular crystallography; diffraction data collected at beamline 19-ID of the Advanced Photon Source at Argonne National Laboratory Structural Biology Center.
In the 1960s and 1970s, scientists observed that plants used one pathway, known as arogenate dehydrogenase (ADH), to make tyrosine. They found that the legume family (i.e., peas, beans, and peanuts), however, had uniquely added a second, called prephenate dehydrogenase (PDH), previously known to be only in microbes. Two years ago, part of this team discovered the genes responsible for making tyrosine, learning that before peanuts and peas evolved into separate lineages, the legumes had evolved PDH enzymes from their existing ADH ones. These sister enzymes are very similar, so only a small number of changes could account for how ADH enzymes evolved into PDH ones. But there were still too many changes to test. The international collaborating teams purified the PDH enzyme of the soybean legume and determined its three-dimensional (3D) structure, which revealed that only a couple of mutations had occurred at the site where the chemical reactions take place. Instead of dozens of possible mutations, there were only two. Thus, changing a single amino acid in the center of the enzyme largely converted the soybean PDH enzyme back into its ancestor ADH enzyme, and this crucial switch also worked in reverse and for enzymes from multiple species. The scientists believe the legume PDH insensitivity to tyrosine could help to produce more tyrosine, and its useful derivatives, in systems like yeast or engineered plants.
Schenck, C. A., et al. “Molecular Basis of the Evolution of Alternative Tyrosine Biosynthetic Routes in Plants,” Nat. Chem. Biol. 13, 1029–1035 (2017). [DOI:10.1038/nchembio.2414].
Instruments and Facilities Used: Structural Biology Center (SBC)–CAT beam 19-ID of the Advanced Photon Source at Argonne National Laboratory.
Scientists are providing the clearest view yet of an intact bacterial microcompartment, revealing the structure and assembly of the organelle’s protein shell at atomic-level resolution. They studied the “photogenic” organelle shell of an ocean-dwelling slime bacteria Haliangium ochraceum. Providing the first view of the shell of an intact bacterial organelle membrane, this full structural view can help provide important information for beneficial use in fighting pathogens or bioengineering bacterial organelles. The research team said these organelles, or bacterial microcompartments (BMCs), are used by some bacteria to fix carbon dioxide. Thus, understanding how the microcompartment membrane is assembled, as well as how it lets some compounds pass through while impeding others, could contribute to research in enhancing carbon fixation and, more broadly, bioenergy. This class of organelles also helps many types of pathogenic bacteria metabolize compounds that are not available to normal, nonpathogenic microbes, giving the pathogens a competitive advantage.
Sutter, M., et al. “Assembly Principles and Structure of a 6.5-MDa Bacterial Microcompartment Shell.” Science 23(6344), 1293–1297 (2017). [DOI:10.1126/science.aan3289].
Instruments and Facilities Used: Michigan State University–DOE Plant Research Laboratory and the Molecular Biophysics and Integrated Bioimaging Division at Lawrence Berkeley National Laboratory; Stanford Synchrotron Radiation Lightsource.
Moissl-Eichinger, C., et al. “Human Age and Skin Physiology Shape Diversity and Abundance of Archaea on Skin.” Sci. Rep. 7, 4039 (2017). [DOI:10.1038/s41598-017-04197-4].
Instruments and Facilities Used: Fluorescence in situ hybridization (FISH); quantitative polymerase chain reaction (PCR); next-generation sequencing; Fourier Transform infrared (FTIR) focal plan array (FPA) hyperspectral imaging. Facilities: Advanced Light Source at the Berkeley Synchrotron Infrared Structural Biology Imaging Project; BioTechMed-Graz, the Bavaria California Technology Center.
Herrmann, J., et al. “Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer,” Biophys. J. 112(9), 1841–1851 (2017). [DOI:10.1016/j.bpj.2017.04.003].
Instruments and Facilities Used: Stanford ChEM-H Macromolecular Structure Knowledge Center, Stanford Department of Structural Biology Electron Microscopy Center, Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory (SLAC). Beamlines or instruments used: transmission electron microscopy (TEM) and small angle X-ray scattering and diffraction (SAXS/D) at SSRL beamline 4-2 at SLAC.
A protein used by common soil bacteria is providing new clues in the effort to convert aryl compounds, a common waste product from industrial and agricultural practices, into something of value. The protein structure of the enzyme LigM was determined using X-ray crystallography, revealing novel structural elements (red in figure) and a conserved tetrahydrofolate-binding domain (gray), with LigM binding to its substrates (green) using internal binding cavities.
Researchers used have resolved the soil bacterium Sphingomonas to metabolize aryl compounds derived from lignin, the stiff, organic material that gives plants their structure. In biofuel production, aryl compounds are a byproduct of the breakdown of lignin, some pathways of which involve demethylation, an often critical precursor to additional modification steps of lignin-derived aryl compounds. The simple, single-enzyme system of LigM, as well as its functionality over a broad temperature range, makes it an attractive demethylase for use in aromatic conversion. Other findings included: half the LigM enzyme was homologous to known structures with a tetrahydrofolate-binding domain that is found in both simple and complex organisms; the other half of LigM’s structure is completely unique, providing a starting point for determining where its aryl substrate-binding site is located; and LigM is a tyrosine-dependent demethylase. This research provides groundwork needed to aid in developing an enzyme-based system for converting aromatic waste into useful products.
Kohlera, A. C., et al. “Structure of Aryl O-Demethylase Offers Molecular Insight into a Catalytic Tyrosine-Dependent Mechanism.” PNAS 114(16), E3205–E3214 (2017). [DOI:10.1073/pnas.1619263114].
Instruments and Facilities Used: Beam line 8.2.2 and X-ray macromolecular crystallography at Berkeley Center for Structural Biology Advanced Light Source at Lawrence Berkeley National Laboratory.
Roth, M. S., et al.. “Chromosome-Level Genome Assembly and Transcriptome of the Green Alga Chromochloris zofingiensis Illuminates Astaxanthin Production.” PNAS 114(21), E4296–E4305 (2017). [DOI:10.1073/pnas.1619928114].
Instruments and Facilities Used: Soft X-ray tomography at National Center for X-ray Tomography (NCXT), operated jointly by Berkeley Lab (LBNL) and University of California, San Francisco, at LBNL’s Advanced Light Source. Other techniques: whole-genome optical mapping, high light RNA sequencing, transcriptome sequencing, and long read sequencing.
Anunciado, D. B., et al. “In Vivo Protein Dynamics on the Nanometer Length Scale and Nanosecond Time Scale.” J. Phys. Chem. Lett. 8(8), 1899–1904 (2017). [DOI:10.1021/acs.jpclett.7b00399].
Instruments and Facilities Used: Quasi-elastic neutron scattering, BASIS neutron backscattering spectrometer at Spallation Neutron Source at Oak Ridge National Laboratory.
In the Ebola virus one of the proteins, VP40, has the ability to refold to achieve new functions; three structural forms of VP40 have been determined, with each structure conferring a separate and essential function in the virus life cycle. The image illustrates (top) a butterfly-shaped dimer critical for membrane trafficking; (middle) a rearranged hexameric structure essential for building and releasing nascent virions; (bottom) an RNA-binding octameric ring that controls transcription in infected cells
O’Dell, W. B., et al. “Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.” Angew. Chem. Int. Ed. 129(3), 785–788 (2017). [DOI:10.1002/anie.201610502].
Instruments and Facilities Used: Neutron crystallography. Joint X-ray/neutron refinement at Center for Structural Molecular Biology at Oak Ridge National Laboratory (ORNL). Diffraction data were collected at SER-CAT 22-ID at the Advanced Photon Source at Argonne National Laboratory and at CG-4D IMAGINE (NSF MRI 09229719) at the High Flux Isotope Reactor at ORNL.
From past studies, scientists think that the molecule TREM2 possibly is involved in cognitive decline, the hallmark of neurodegenerative diseases, because certain mutations that alter the structure of TREM2 are associated with an increased risk of developing late-onset Alzheimer’s, frontal temporal dementia, Parkinson’s disease, and sporadic amyotrophic lateral sclerosis (ALS). Other TREM2 mutations are linked to Nasu-Hakola disease, a rare inherited condition that causes progressive dementia and death in most patients by age 50.Although its exact contribution is unknown, dysfunctional TREM2 does relate to neurodegeneration, and “inflammation is the common thread in all these conditions.” The scientists investigated what TREM2 mutations do to the structure of the protein itself to impact its function, so ways can be found to correct it.
Structural analysis of TREM2 revealed that the mutations associated with Alzheimer’s alter the surface of the protein, while those linked to Nasu-Hakola influence the “guts” of the protein. The difference in location could explain the severity of Nasu-Hakula, in which signs of dementia begin in young adulthood. The internal mutations totally disrupt the structure of TREM2, resulting in fewer TREM2 molecules. The surface mutations, in contrast, leave TREM2 intact but likely make it harder for the molecule to connect to proteins or send signals as normal TREM2 molecules would.
TREM2 lies on the surface of immune cells called microglia, which are thought to be important “housekeeping” cells involved in, for example, maintaining healthy brain biology via a process called phagocytosis that cleans cellular waste, including the amyloid beta that is known to accumulate in Alzheimer’s disease. If the microglia lack TREM2 or the TREM2 is dysfunctional, these cellular housekeepers can’t perform their cleanup tasks. Though the exact function of TREM2 is still unknown, mice without TREM2 have defects in their microglia. With these structural data, scientists can study how TREM2 works, or doesn’t work, in these neurodegenerative diseases, as well as other inflammatory conditions including chronic obstructive pulmonary disease and stroke. The structure of TREM2 could be important for understanding many chronic and degenerative diseases.
Kober, D. L., et al. “Neurodegenerative Disease Mutations in TREM2 Reveal a Functional Surface and Distinct Loss-of-Function Mechanisms,” eLife 5, e20391 (2016). [DOI:10.7554/eLife.20391].
Instruments and Facilities Used: X-ray Facility at the Advanced Photon Source (APS) at Argonne National Laboratory
The flexibility of this macromolecule suggests that it could be useful for the controlled capture and release of smaller compounds. Overall, the researchers expect that, with the tools and techniques developed here, the combination of SAXS with crystallography or electron microscopy could be increasingly useful in analyzing and optimizing designed protein assemblies and understanding their behavior in solution.
Lai, Y.-T., et al. “Designing and Defining Dynamic Protein Cage Nanoassemblies in Solution.” Sci. Adv. 2(12), e1501855 (2016). [DOI:10.1126/sciadv.1501855].
Instruments and Facilities Used: Small-angle X-ray scattering (SAXS) at Advanced Light Source (ALS) with SIBYLS Beamline 12.3.1 (a joint crystallography and SAXS beamline) at Lawrence Berkeley National Laboratory.
The crystal structure of NOV1, a stilbene cleaving oxygenase, shows the features of this enzyme at atomic resolution. This protein fold view highlights the placement of an iron (orange), dioxygen (red), and resveratrol, a representative substrate (blue) in the active site of the enzyme. Enzymes such as NOV1 could be of value in the biological production of important molecular fragments derived from lignin. (Image courtesy of Ryan McAndrew/JBEI and Berkeley Lab)
McAndrew, R.P., N. Sathitsuksanoh, M.M. Mbughuni, R.A. Heins, J.H. Pereira, A. George, K.L. Sale, B.G. Fox, B.A. Simmons, and Paul D. Adams. 2016. “Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase” PNAS 113 (50) 14324-14329. doi:10.1073/pnas.1608917113
A strategy was developed that designs interfaces onto idealized proteins aimed to direct their assembly into multimeric complexes. Researchers used structural characterization, both X-ray crystallography and small angle X-ray scattering (SAXS), to show that many of the designs adopted the target oligomerization state and predicted structure. Not only does the work demonstrate that scientists have a basic understanding of what determines oligomerization, but that they could design proteins with tunable shape, size, and symmetry for a variety of biological applications.
Fallas, J. A., et al. “Computational Design of Self-Assembling Cyclic Protein Homo-Oligomers.” Nat. Chem. 9, 353–360 (2017). [DOI:10.1038/nchem.2673].
Instruments and Facilities Used: Crystallography Collective program; beamline 5.0.2; small angle X-ray scattering (SAXS) on the SIBYLS BL12.3.1 beamline. Advanced Photon Source synchrotron beamline 24-ID-C at Berkeley Center for Structural Biology and Advanced Light Source at Lawrence Berkeley National Laboratory (LBNL).
Understanding the logic of nuclear architecture and how it contributes to stem-cell differentiation remains challenging. Until now, imaging the nucleus was possible only indirectly, by staining it, hopefully with even distribution. In this work, researchers used soft X-ray microtomography to record a series of images from mouse olfactory nerve cells in three separate stages of development. This unique technique provides a new way of looking at the nucleus without the need to chemically treat the cell, allowing visualization of intact cells in a near-native state at a resolution of about 50 nanometers.
After imaging frozen cells at each stage from dozens of different angles, the researchers used each set of 2D images to calculate a 3D reconstruction detailing the changing chromatin formations in the nucleus, collecting the images using soft X-rays within the “water window” [i.e., 284 to 543 electron electronvolts (eV)]. The absorption of X-rays by biomolecules is linear with biochemical composition and concentration, generating a unique X-ray linear absorption coefficient measurement for each voxel (3D pixel analog). Thus, the researchers were able to visually distinguish between different types of chromatin—heterochromatin, due to an increased biomolecular concentration, appears darker than euchromatin in computer-generated tomographic orthoslices (virtual sections) through the nucleus.
The results showed that chromatin compaction increases as the cell matures, and that condensed chromatin moves to the nuclear core during differentiation. Though the chromatin was thought to exist as a series of disconnected islands, the results showed how it was compartmentalized into two distinct regions of “crowding” that form a continuous network throughout the nucleus. Also, based on comparison of these results to those of similar cells in which the gene for a heterochromatin binding protein had been inactivated (“knocked out”), the researchers concluded that it regulates the reorganization of chromatin in mature neurons.
Soft x-ray tomography provides a powerful method to study chromatin and nuclear architecture in vivo. There is no need for chemical fixation and sectioning, preventing a plethora of artifacts introduced either by fixatives or by visualization of only thin sections. The researchers believe performing statistical analyses is now possible to based on large collections of cell nuclei images sorted by different stages of development.
Le Gros, M. A., et al. “Soft X-Ray Tomography Reveals Gradual Chromatin Compaction and Reorganization During Neurogenesis In Vivo,” Cell Reports 17(8), 2125–2136 (2016). [DOI:10.1016/j.celrep.2016.10.060].
Instruments and Facilities Used: Soft X-ray tomography (beamline 2.1) of the Advanced Light Source at the National Center for X-Ray Tomography at Lawrence Berkeley National Laboratory.
Ptychography is a coherent diffractive imaging technique with high resolution (7 nm in this work) and excellent chemical-state sensitivity. Researchers obtained ptychographic absorption and phase spectra of magnetosomes from a marine MTB (Magnetovibrio blakemorei strain MV-1). The data, obtained from mature magnetosomes, immature magnetosomes, precursor regions, and the gaps between magnetosome chains, indicated that different iron species can coexist in a single cell. Based on the results, the researchers proposed a model in which soluble Fe(II) is taken up from the environment and partially oxidized to Fe(III), which in turn is then oxidized and transformed into hematite and ultimately into magnetite.
Researchers also used ptychography to measure the X-ray magnetic circular dichroism (XMCD) spectra of both extracellular and intracellular magnetosomes. XMCD probes the magnetism of a crystal in a site-specific manner, showing that absorption and XMCD ptychography signals provide complementary information. These experiments demonstrate that ptychography, which combines high spatial resolution, high-sensitivity chemical speciation, and site-specific magnetic information, offers a powerful probe for biomineralization studies.
Zhu, X., et al. “Measuring Spectroscopy and Magnetism of Extracted and Intracellular Magnetosomes Using Soft X-Ray Ptychography,” PNAS 113, E8219 (2016). [DOI:10.1073/pnas.1610260114].
Instruments and Facilities Used: Scanning transmission X-ray microscope (STXM) on beamline 10ID1 of Canadian Light Source at University of Saskatchewan and the Advanced Light Source Beamlines 126.96.36.199 and 11.0.2 at Lawrence Berkeley National Laboratory.
Perticaroli, S., et al. “Description of Hydration Water in Protein (Green Fluorescent Protein) Solution.” J. Am. Chem. Soc. 139(3), 1098–1105 (2017). [DOI:10.1021/jacs.6b08845].
Instruments and Facilities Used: Neutron scattering at Center for Structural Molecular Biology and Spallation Neutron Source at Oak Ridge National Laboratory.
Zinc is carried through the body by a protein known as serum albumin. Scientists proved the location of the primary site where serum albumin binds with zinc, but the team also found several more secondary binding sites, revealing a more complex interaction than anticipated.
While computer models previously had been used to predict how serum albumin picks up zinc, the team used X-ray macromolecular crystallography to obtain data-based images of protein crystals showing how zinc actually bound to serum albumin. The technique allowed them to pinpoint the location of each particular zinc atom, though it was a challenging task. The schematics produced allow scientists to see, for the first time, exactly how serum albumin and zinc come together. Serum albumin also transports many other things, such as hormones and fatty acids.
Too much zinc is toxic to the body, so it must make zinc available where it is needed, but prevent harmful, excessive buildup. The study’s findings could help shed light on why certain drugs affect some patients differently than others.
Handing, K. B., et al. “Circulatory Zinc Transport is Controlled by Distinct Interdomain Sites on Mammalian Albumins.” Chem. Sci. 7, 6635 (2016). [DOI:10.1039/c6sc02267g].
Instruments and Facilities Used: X-ray macromolecular crystallography; Life Sciences Collaborative Access Team (LS-CAT) 21-ID-F and 21-ID-G X-ray beamlines and Structural Biology Center (SBC)-CAT 19-BM-D X-ray beamline, all at the Advanced Photon Source at Argonne National Laboratory
The plant cellulose synthesis complex is a large multi-subunit transmembrane protein complex responsible for synthesis of cellulose chains and their assembly into microfibrils. The image shows ab initio structures of CESA trimers calculated from small-angle scattering data represented by semi-transparent grey surface envelopes, superposed with the computational atomic models in orange. Image credits: Thomas Splettstoesser, scistyle.com, Berlin Germany
By pairing high-resolution chemical imaging analysis with a long-term litter decomposition experiment under field conditions, the researchers discovered that litter decomposition is tightly coupled to redox cycling of Mn. The researchers discovered why Mn exerts such a strong control on litter decomposition rates and the mechanism of the underlying biogeochemical process, observing molecular changes within the litter as it decomposed over the years and probing structural changes within the very large biomolecules present in litter that frequently are not directly detectable by other means. This tunable technique also allowed the scientists to selectively ionize different biopolymers present in the litter, such as lignin, which is an important biopolymer because it lends rigidity to plant cell walls and protects litter from microbial decay.
In microenvironments within the litter, microbes actively cycle Mn as they colonize and break down the litter, and research indicates that biogeochemical constraints on bioavailability, mobility, and Mn reactivity in the plant-soil system may have a profound impact on litter decomposition rates. Understanding more about the mobility and reactivity of Mn in the plant-soil system also helps researchers improve their ability to accurately predict carbon cycling trends in ecosystems, contributing to greater insights into climate change patterns.
Keiluweit, M.. et al. “Long-Term Litter Decomposition Controlled by Manganese Redox Cycling,” PNAS 12(38), E5253–E5260 (2015). [DOI:10.1073/pnas.1508945112].
Instruments and Facilities Used: Beamlines 10.3.2 (X-ray microprobe capabilities) and 1.4.3 (Fourier transform infrared spectromicroscopy) at Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory; beamline 4-3 at Stanford Synchrotron Radiation Light Source (SSRLS).
The spherical protein ferritin was used to synthesize a porous 3-D crystalline framework material, and further engineered to have metal hubs on its surface. Organic molecules bridge these hubs, and in a controlled way create a porous material with potential uses from efficient fuel storage to carbon capture and conversion. The study shows the great potential for proteins as building blocks with exquisite properties of electron transfer and bioinspired catalysis.
A. Sontz, J. B. Bailey, S. Ahn and F. A. Tezcan, “A Metal Organic Framework with Spherical Protein Nodes: Rational Chemical Design of 3D Protein Crystals”, J. Am. Chem. Soc. 137, 11598 (2015), doi: 10.1021/jacs.5b07463
Nitrogen fixation is required for all forms of life, being essential for the biosynthesis of molecules that are used in creating plants and organisms. Nitrogenase is the only known enzyme capable of performing this multi-electron reduction, and understanding how it does this conversion is of high importance also for the production of ammonia (as fertilizer), for energy efficiency (as industrial processes to produce ammonia consumes enormous amounts of energy), and for global warming (capturing N2). The structure of the CO inhibitor bound to the FeMo-cofactor active site in nitrogenase at high resolution provides insight into a catalytic competent state, establishes the importance of a bridging S atom, and indicates how N2 might bind during turn-over. Stanford Synchrotron Radiation Lightsource.
Spatzal, K.A. Perez, O. Einsle, J.B. Howard, D.C. Rees, “Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase” Science 345, 1620-1623 (2014), doi: 10.1126/science.1256679
Illustration of structural rearrangement of cellulose and matrix copolymers during thermochemical pretreatment of lignocellulose biomass. Image credits: Thomas Splettstoesser, scistyle.com, Berlin Germany