Caroline Ajo-Franklin, a staff scientist in the Biological Nanostructures Facility at Lawrence Berkeley National Laboratory’s (LBNL) Molecular Foundry (one of the Nanoscale Science Research Centers supported by DOE’s Office of Basic Energy Sciences), teamed up with Corie Ralston to use X-ray mass spectrometry footprinting at LBNL’s Advanced Light Source. Ralston, who works in the Molecular Biophysics and Integrated Bioimaging (MBIB) Division, uses the X-ray mass spectrometry footprinting technique to precisely probe proteins and their surroundings at the Advanced Light Source. Ajo-Franklin and Ralston saw that they could use footprinting to answer a long-standing question in microbiology: how do bacterial proteins interact directly with minerals to transfer electrons and allow the microbe to live?
“Understanding what these interactions between proteins and materials look like can help us design them better,” Ajo-Franklin said, “and give us insight on how to connect living cells with devices.”
Surprisingly, “the biggest finding … was that our proteins bind relatively weakly,” Ajo-Franklin noted. “Most proteins that interface with materials bind really tightly,” changing shape as they form the connection. This particular protein does not appear to change shape at all and only interacts with the mineral in a small area, requiring about five times less binding energy, by comparison, than typical proteins that form biominerals. This finding makes a lot of sense, Ajo-Franklin, because this protein’s job “is to transfer electrons to the mineral, so it doesn’t have to be in contact for very long.”
To characterize microbes on human skin, researchers from Austria, including Christine Moissl-Eichinger, collaborated with Hoi-Ying Holman and other scientists at the Berkeley Synchrotron Infrared Structural Biology Imaging Program, a BER-supported infrared beamline at the Advanced Light Source at Lawrence Berkeley National Laboratory. The research stemmed from a joint project between the National Aeronautics and Space Administration and the European Space Agency.
“We were checking spacecraft and their clean rooms for the presence of archaea, as they are suspected to be possible critical contaminants during space exploration,” Moissl-Eichinger said. “Certain methane-producing archaea, the so-called methanogens, could possibly survive on Mars. We did not find many signatures from methanogens, but we found loads of Thaumarchaeota, a very different type of archaea that survives with oxygen.”
This finding led to the discovery that these archaea are present on people’s skin. The infrared beamline was used to rapidly and precisely characterize samples from humans and determine the levels and types of microbes present, based on the chemical specificity of infrared spectroscopy. This analysis could then be linked back to the genomic data collected by the Austrian team. The detected archaea are probably involved in nitrogen turnover on skin and are capable of lowering skin pH, supporting the suppression of pathogens. The researchers found that because of changes in skin moisture, these microbes are most abundant in subjects younger than 12 and older than 60.
Reaction centers (RCs) are pigment-protein complexes that drive photosynthesis by converting light into chemical energy (electrons) to power the cell, possibly arising from a homodimeric protein. The symmetry of a homodimer is broken in heterodimeric rRC structures, while the 2.2-Angstrom resolution X-ray structure of the homodimeric RC-photosystem from the phototroph Heliobacterium modesticaldum exhibits perfect C2 symmetry. The core polypeptide dimer and two small subunits coordinate 54 bacteriochlorophylls and 2 carotenoids that capture and transfer energy to the electron-transfer chain at the center, which performs charge separation and comprises 6 (bacterio)chlorophylls and an iron-sulfur cluster; unlike other RCs, it lacks a bound quinone. This structure preserves characteristics of the ancestral reaction center, providing insight into the evolution of photosynthesis.
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.
Funding Acknowledgements: Funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, through grant DE-SC0010575 to K.E.R., R.F., and J.H.G.; supported with x-ray crystallographic equipment and infrastructure provided by P. Fromme of the Biodesign Center for Applied Structural Discovery at Arizona State University. Berkeley Center for Structural Biology supported in part by the National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS) and the Howard Hughes Medical Institute (HHMI). The Advanced Light Source (ALS) is a DOE Scientific User Facility (SUF) supported by the Director, OBES, DOE Office of Science, and operated for DOE Office of Science by Lawrence Berkeley National Laboratory (LBNL). Results derived from work performed at Argonne National Laboratory’s (ANL) Structural Biology Center (SBC) at the Advanced Photon Source (APS). SBC funded by the Office of Biological and Environmental Research (BER), DOE Office of Science. ANL is operated by University of Chicago Argonne, LLC, for the DOE Office of Science under contract DE-AC02-06CH11357.
The simplest known bacterium able to drive photosynthesis is found in muddy soils near hot springs. Heliobacterium modesticaldum is a sun-loving, soil-dwelling, thermophilic bacterium that photosynthesizes near-infrared light, unlike plants, which use different parts of visible light. The photosynthesis reaction centers (RCs) of H. modesticaldum are thought to resemble the earliest common ancestor of all photosynthesis complexes, which evolved around three billion years ago. Thus, a clear, detailed picture of the H. modesticaldum RC would provide valuable insight into the early evolution of photosynthesis. However, successfully purifying an RC protein and growing the crystals needed for X-ray crystallography can be a lengthy, difficult process.
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.” Science357(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.
Funding Acknowledgements: Work funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, through grant DE-SC0010575 to K.E.R., R.F., and J.H.G. and supported with x-ray crystallographic equipment and infrastructure provided by P. Fromme of the Biodesign Center for Applied Structural Discovery at Arizona State University. Berkeley Center for Structural Biology supported in part by National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS) and Howard Hughes Medical Institute (HHMI). The Advanced Light Source (ALS) is a DOE Scientific User Facility supported by OBES, Director, DOE Office of Science, and operated for DOE Office of Science by Lawrence Berkeley National Laboratory (LBNL). Results derived from work performed at Argonne National Laboratory’s (ANL) Structural Biology Center (SBC) at the Advanced Photon Source (APS). SBC funded by the Office of Biological and Environmental Research (OBER), DOE Office of Science. ANL is operated by University of Chicago Argonne, LLC, for the DOE Office of Science under contract DE-AC02-06CH11357.
Scientists have discovered the details of an unconventional coupling between a bacterial protein and a mineral that allows the bacterium to breathe when oxygen is not available. This research could lead to new innovations in linking proteins to other materials for bio-based electronic devices, such as sensors that can diagnose disease or detect contaminants. It also could help researchers to understand and control the chemical reactions sparked by these protein-material interactions and, eventually, how organisms remodel their environment and make biominerals.
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.
Funding Acknowledgements: Work supported by the Laboratory Directed Research and Development Program of Lawrence Berkeley National Laboratory (LBNL), performed at the Molecular Foundry and Advanced Photon Source (APS) at Argonne National Laboratory (ANL) and used resources of the Joint BioEnergy Institute (JBEI), supported by the Office of Basic Energy Sciences (OBES) and Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science, under Contract No. DE-AC02-05CH11231. CMA-F support: Office of Naval Research, Award number N000141310551.
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,” Science357(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.
Funding Acknowledgements: Advanced Light Source (ALS) 8.3.1 beamline, Lawrence Berkeley National Laboratory (LBNL), and Stanford Synchrotron Radiation Lightsource (SSRL) 9-2 beamline, SLAC National Accelerator Laboratory (SLAC), for assistance with data collection. ALS Beamline 8.3.1, is operated by University of California Office of the President, Multicampus Research Programs and Initiatives (grant MR-15-328599), and Program for Breakthrough Biomedical Research, partially funded by the Sandler Foundation. Use of SSRL supported by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under contract no. DE-AC02-76SF00515. Electron microscopy (EM) data collected in Howard Hughes Medical Institute (HHMI) EM facility located at University of California, Berkeley. SSRL Structural Molecular Biology Program supported by DOE Office of Biological and Environmental Research (OBER) and the National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; including grant no. P41GM103393). Project funded by U.S. National Science Foundation (NSF) grant no. 1244557 (to J.A.D.) and NIGMS grant no. 1P50GM102706-01 (to J. H. Cate). A.V.W. and K.W.D. support: NSF Graduate Research Fellowship; G.J.K. funding: HHMI. J.A.D. and E.N.: HHMI investigators and members of the Center for RNA Systems Biology. Atomic coordinates and structure factors for the reported crystal structures deposited in the Protein Data Bank under accession codes 5VVJ (half-site–bound), 5VVK (pseudo–full-site–bound), and 5VVL (pseudo–full-site–bound with Ni2+). Cryo-EM structure and map deposited in the Protein Data Bank under accession code 5WFE and the Electron Microscopy Data Bank under accession code EMD-8827.
A research study has identified and characterized a new, functionally distinct member of the orange carotenoid protein (OCP) family. The OCP complex enables chromatically acclimating blue-green algae to avoid cellular damage and growth inhibition in conditions of high light or nutrient stress. In a recent bioinformatic analysis of all available cyanobacterial genomes, the group found that many of these ecophysiologically diverse organisms encode more than one copy of the full-length OCP. The study’s focus was the filamentous blue-green algae Tolypothrix, which encodes two OCPs.
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 Plants3, 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.
Funding Acknowledgements: Work supported by the National Science Foundation (NSF; IOS 1557324). Advanced Light Source supported by the Office of Basic Energy Sciences (OBES), Director, U.S. Department of Energy (DOE) Office of Science, under Contract No. DE-AC02-05CH11231.
Structure of microcompartment’s protein shell could help research in bioenergy, pathogenesis, and biotechnology
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.” Science23(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.
Funding Acknowledgements: Support: National Institutes of Health’s (NIH) National Institute of Allergy and Infectious Diseases (NIAID) grant 1R01AI114975-01 and the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under contract no. DE-FG02-91ER20021. Advanced Light Source (ALS), Lawrence Berkeley National Laboratory (LBNL) support: OBES, Director, DOE Office of Science, under contract no. DE-AC02-05CH11231. B.G. support: advanced postdoctoral mobility fellowship from Swiss National Science Foundation (NSF; project P300PA_160983). Use of Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory (SLAC) support: OBES, DOE Office of Science, under contract no. DE-AC02-76SF00515. M.S. and C.A.K.: inventors on patent application 62509553 submitted by LBNL that covers strategies for scaling the shell-protein system described in this work. Cryo-EM map of complete shell deposited at Electron Microscopy Data Bank (EMDB) with accession code EMD-8747. X-ray crystallographic coordinates and structure-factor files deposited in Protein Data Bank (PDB) under the following accession numbers: 5V74 (complete shell), 5V75 (BMC-T2), and 5V76 (BMC-T3).
What’s on your skin? Your skin is crawling with single-celled microorganisms—and they’re not just bacteria. This study found that the skin microbiome also contains the extreme-loving archaea, and that the amount of these microbes varies with age. The microbiome forms a protective layer that protects the body from pathogens. Both genetic and chemical analyses of samples collected from human volunteers ranging in age from 1 to 75 showed that archaea were most abundant in subjects younger than 12 and older than 60. Before the study, the existence of archaea on human skin was unknown, but they are now known to play an important part. The international study also determined that gender was not a factor but that people with dry skin have more archaea. Results from genetic analysis and infrared spectroscopy imaging allowed scientists to link lower levels of oily secretion of sebaceous glands and with increased archaea, most of which have proven to be beneficial.
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.
Funding Acknowledgements: Support: BioTechMed-Graz, the Bavaria California Technology Center (BaCaTeC), and University of Regensburg. AJP support: German National Academic Foundation (Studienstiftung des deutschen Volkes). Infrared (IR) support: Berkeley Synchrotron Infrared Structural Biology (BSISB) Program, Lawrence Berkeley National Laboratory (LBNL), funded by the Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science. Advanced Light Source (ALS) at LBNL support: Office of Basic Energy Sciences (OBES), Director, DOE Office of Science, through Contract DE-AC02-225 05CH11231.
Joint BioEnergy Institute study targets LigM for its role in breaking down aromatic pollutants
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.” PNAS114(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.
Funding Acknowledgements: Crystallographic work: Berkeley Center for Structural Biology (BCSB) Advanced Light Source (ALS) beam line 8.2.2. BCSB ALS staff: technical support; J. H. Pereira: assistance in early stages of crystallographic work. BCSB support in part: National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS) and Howard Hughes Medical Institute (HHMI). ALS support: Office of Basic Energy Sciences (OBES), Director, U.S. Department of Energy (DOE) Office of Science, under Contract DE-AC02-05CH11231. Work conducted by Joint BioEnergy Institute (JBEI) and supported by Office of Biological and Environmental Research (OBER), DOE Office of Science, under Contract DE-AC02-05CH11231.