Summary
Growth of Methylacidiphilum fumariolicum SolV, an extremely acidophilic methanotrophic microbe isolated from an Italian volcanic mudpot, is shown to be strictly dependent on the presence of ...lanthanides, a group of rare earth elements (REEs) such as lanthanum (Ln), cerium (Ce), praseodymium (Pr) and neodymium (Nd). After fractionation of the bacterial cells and crystallization of the methanol dehydrogenase (MDH), it was shown that lanthanides were essential as cofactor in a homodimeric MDH comparable with one of the MDHs of Methylobacterium extorquens AM1. We hypothesize that the lanthanides provide superior catalytic properties to pyrroloquinoline quinone (PQQ)‐dependent MDH, which is a key enzyme for both methanotrophs and methylotrophs. Thus far, all isolated MxaF‐type MDHs contain calcium as a catalytic cofactor. The gene encoding the MDH of strain SolV was identified to be a xoxF‐ortholog, phylogenetically closely related to mxaF. Analysis of the protein structure and alignment of amino acids showed potential REE‐binding motifs in XoxF enzymes of many methylotrophs, suggesting that these may also be lanthanide‐dependent MDHs. Our findings will have major environmental implications as metagenome studies showed (lanthanide‐containing) XoxF‐type MDH is much more prominent in nature than MxaF‐type enzymes.
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal ...disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
A new approach to the cosmological recombination problem is presented, which completes our previous analysis on the effects of two-photon processes during the epoch of cosmological hydrogen ...recombination, accounting for ns-1s and nd-1s Raman events and two-photon transitions from levels with n≥ 2. The recombination problem for hydrogen is described using an effective 400-shell multilevel approach to which we subsequently add all important recombination corrections discussed in the literature thus far. We explicitly solve the radiative transfer equation of the Lyman-series photon field to obtain the required modifications to the rate equations of the resolved levels. In agreement with earlier computations, we find that 2s-1s Raman scattering leads to a delay in recombination by ΔN
e/N
e∼ 0.9 per cent at z∼ 920. Two-photon decay and the Raman scattering from higher levels (n > 3) result in small additional modifications, and precise results can be obtained when including their effect for the first three to five shells. This work is a major step towards a new cosmological recombination code (cosmorec) that supersedes the physical model included in recfast, and which, owing to its short run time, can be used in the analysis of future cosmic microwave background data from the PLANCK Surveyor.
The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in ...various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.
X-ray free-electron lasers (XFELs) enable crystallographic structure determination beyond the limitations imposed upon synchrotron measurements by radiation damage. The need for very short XFEL ...pulses is relieved through gating of Bragg diffraction by loss of crystalline order as damage progresses, but not if ionization events are spatially non-uniform due to underlying elemental distributions, as in biological samples. Indeed, correlated movements of iron and sulfur ions were observed in XFEL-irradiated ferredoxin microcrystals using unusually long pulses of 80 fs. Here, we report a femtosecond time-resolved X-ray pump/X-ray probe experiment on protein nanocrystals. We observe changes in the protein backbone and aromatic residues as well as disulfide bridges. Simulations show that the latter's correlated structural dynamics are much slower than expected for the predicted high atomic charge states due to significant impact of ion caging and plasma electron screening. This indicates that dense-environment effects can strongly affect local radiation damage-induced structural dynamics.
The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which ...allow data collection from nanometre- to micrometre-sized crystals in a 'diffraction-before-destruction' approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins.
Results from the Kepler mission indicate that the occurrence rate of small planets (<3 R⊕) in the habitable zone of nearby low-mass stars may be as high as 80%. Despite this abundance, probing the ...conditions and atmospheric properties on any habitable-zone planet is extremely difficult and has remained elusive to date. Here, we report the detection of water vapor and the likely presence of liquid and icy water clouds in the atmosphere of the 2.6 R⊕ habitable-zone planet K2-18b. The simultaneous detection of water vapor and clouds in the mid-atmosphere of K2-18b is particularly intriguing because K2-18b receives virtually the same amount of total insolation from its host star ( 1368 − 107 + 114 W m−2) as the Earth receives from the Sun (1361 W m−2), resulting in the right conditions for water vapor to condense and explain the detected clouds. In this study we observed nine transits of K2-18b using Hubble Space Telescope/WFC3 in order to achieve the necessary sensitivity to detect the water vapor, and we supplement this data set with Spitzer and K2 observations to obtain a broader wavelength coverage. While the thick hydrogen-dominated envelope we detect on K2-18b means that the planet is not a true Earth analog, our observations demonstrate that low-mass habitable-zone planets with the right conditions for liquid water are accessible with state-of-the-art telescopes.
The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast ...structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together with the observed oscillations of residues contacting the heme, our calculations support the prediction that an immediate collective response of the protein occurs upon ligand dissociation, as a result of heme vibrational modes coupling to global modes of the protein.
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