Exploring the Roots of Photosynthesis in a Soil-Dwelling Bacterium

structure H. modesticaldum
Overall structure of the H. modesticaldum photosynthetic reaction center. The transmembrane helices are shown in gold and red. Light-harvesting “antenna” molecules are colored green, and the electron-transfer chain is colored blue. [From Gisriel, C., et al. “Structure of a Symmetric Photosynthetic Reaction Center–Photosystem,” Science 357(6355), 1021–1025 (2017). DOI:10.1126/science.aan5611. Reprinted with permission from AAAS.]
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.” 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.

New Chemical Strategy for Conversion of CO2 to Biomass

acetone carboxylase
Upon nucleotide binding, acetone carboxylase undergoes conformational shifts, opening an internal solvent channel. (a) Ligand-free structure showing a substrate channel (grey) linking the nucleotide binding site to the outside solvent and allowing adenosine triphosphate (ATP) cofactor and carbon dioxide (CO2) substrate to enter. Access to the manganese (Mn) active site is closed by an α-helix in the path. (b) The adenosine monophosphate (AMP)–bound structure shows an opening of an internal channel (grey) linking the nucleotide binding site to the Mn active site. The blocking helix becomes a disordered loop when AMP is bound. [From 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. Reused under a Creative Commons license (CC by 4.0, https://creativecommons.org/licenses/by/4.0/).]
A research team has uncovered a new mechanism for enzyme-mediated carbon dioxide (CO2) capture and conversion. They have revealed new and unique elements of carboxylation chemistry (CC) that could be used in catalytic strategies for converting CO2 into chemical feedstock or biomass. The crystal structure of acetone carboxylase (AC; enzyme involved in biodegradation by bacteria) was solved using X-ray crystallographic data. The inactive, unbound (apo) structure shows a substrate channel blocked off from the manganese (Mn) active site where CO2 conversion takes place. An adenosine monophosphate (AMP)–bound structure contains large conformational changes that open the channel to the active site. Highly reactive intermediates in the channel are protected from outside solvent as they are transported to the Mn active site for CO2 conversion. Stepwise mechanisms of carboxylation reactions differ in essential ways with respect to co-substrate, co-factor, and metal requirements. Knowledge of these mechanisms provides the basis for an increased fundamental understanding of CC, contributing to future strategies for CO2 capture and conversion to biomass. These in turn may mitigate the effects of increasing concentrations of CO2 on the global climate.

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.

The CRISPR Target-Recognition Mechanism

Cas1-Cas2 complexSurface representation of the Cas1-Cas2 complex, consisting of four Cas1 proteins (light and dark green) and two Cas2 proteins (yellow). Donor DNA (brown) is being integrated into the target DNA (blue), at a precise location in the CRISPR array, following a short leader sequence (red). [From Wright, A. V., et al.  “Structures of the CRISPR Genome Integration Complex,” Science 357(6356), 1113–1118 (2017). [DOI:10.1126/science.aao0679. Reprinted with permission from AAAS*.]

* Readers may view, browse, and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes. Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher.



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.


Novel Orange Carotenoid Proteins Shedding Light on Evolution of Cyanobacteria Photoprotection

blue-green algae
Filamentous blue-green algae [Courtesy Landcare Research. Reused under a Creative Commons license (CC By 4.0, https://creativecommons.org/licenses/by/4.0/).]
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 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.

Finding New Clues to a Common Respiratory Virus

protein structure
Illustrated structure of a protein that helps a common respiratory virus evade the immune system. The structure was determined via X-ray crystallography at the Advanced Photon Source at Argonne National Laboratory. [Adapted by permission from Springer Nature: 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. Copyright 2017.]
Each year in the United States, more than 57,000 children younger than 5 years old are hospitalized due to respiratory syncytial virus (RSV) infection, and about 14,000 adults older than 65 die from it. By age 2, most children have been infected with RSV, experiencing usually only mild cold symptoms. People with weakened immune systems, however, such as infants and the elderly, can face serious complications, including pneumonia and, in some cases, death. Now, scientists studying the virus using bright X-rays have found clues to how RSV causes disease. They mapped the molecular structure of an RSV protein that interferes with the body’s ability to fight off the virus. Knowing the structure of this protein will help them understand how the virus impedes immune response, potentially leading to a vaccine or treatment for this common infection.

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.

Pulling the Tablecloth Out from Under Essential Metabolism

tomatoes peanuts soybeans
For the first time, scientists have caught a vital cell machinery molecule in the process of evolving. A key enzyme plants use to make tyrosine, an amino acid necessary for life, was thought to be conserved across the plant kingdom, but the scientists found a mutated form in legumes. In cherry tomatoes, the basic canonical form of the enzyme dominates; peanuts can switch hit; and some strains of soybeans (lumpy beans at right) have lost the canonical form. [Courtesy Jez laboratory, Washington University in St. Louis]
Plants caught in the act, changing chemistry thought to be immutable because necessary for life

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.

Revealing How Bacterial Organelles Assemble

Kerfeld and Sutter

Cheryl Kerfeld and Markus Sutter handle crystallized proteins at Berkeley National Laboratory’s Advanced Light Source. [Image courtesy Marilyn Chung, Lawrence Berkeley National Laboratory]
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.” 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.

Could This Enzyme Help Turn Biofuel Waste into Something Useful?

LigM Structure
The protein structure of LigM was determined using X-ray crystallography, revealing novel structural elements that are unique to LigM (red) in addition to a conserved tetrahydrofolate-binding domain (gray) that is found throughout life. LigM binds to its substrates (green) using internal binding cavities. [From 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.]
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.” 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.

Ebola Virus

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

Bornholdt, T. Noda, D.M. Abelson, P. Halfmann, M.R. Wood, Y. Kawaoka, E. Ollmann Saphire, Cell 154, 763 (2013) [DOI: 10.1016/j.cell.2013.07.015]

Neutrons Identify Oxygen Activation in LPMOs

Fungal lytic polysaccharide monooxygenases (LPMOs)
Scientists used neutrons to probe the structural details of the specialized LPMO fungal enzyme that relies on oxidation to digest molecules, aiming to improve the efficiency of enzymatic cellulose breakdown. [From O’Dell, W. B., P. K. Agarwal, and F. Meilleur. “Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.” Angew. Chem. Int. Ed. 56, 767–770 (2017). [DOI:10.1002/anie.201610502]. ©2017 The Authors. Published by Wiley-VCH Verla GmbH & Co. KGaA, Weinheim.]
Fungal lytic polysaccharide monooxygenases (LPMOs) are known to enhance the efficiency of cellulose-hydrolyzing enzymes through oxidative cleavage of the glycosidic bonds. For this study, PMO-2 from Neurospora crassa was heterologously expressed from Pichia pastoris, purified, and crystallized for high-resolution X-ray crystal structures that revealed “prebound” molecular oxygen in the resting state and a dioxo species in complex with the catalytic copper (Cu2+) ion, which is the first structural description of molecular oxygen (O2) activation by a LPMO. In addition, neutron diffraction studies and density functional theory calculations have identified a role for a conserved histidine in promoting oxygen activation. Extension of these studies to the enzyme-substrate complex could provide a complete picture of the enzymatic mechanism for the potential benefit of applications such as bioethanol production.

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.