Roles of acyl carrier proteins in ladderane fatty acid producing-organisms.

Ladderanes are highly strained hydrocarbons consisting of two or more linearly concatenated cyclobutane rings. Strikingly, ladderane moieties are part of unique fatty acids and fatty alcohols that are exclusively found in the membrane lipids of anaerobic ammonium-oxidizing (anammox) bacteria. These bacteria express a distinctive gene cluster (cluster I) that has been suggested to be responsible for ladderane fatty acid (FA) biosynthesis in addition to a cluster likely involved in canonical FA biosynthesis (cluster III).

Redox potential tuning by calcium ions in a novel c-type cytochrome from an anammox organism.

The electrochemical potentials of redox-active proteins need to be tuned accurately to the correct values for proper biological function. Here, we describe a diheme cytochrome c with high heme redox potentials of about +350 mV, despite having a large overall negative charge, which typically reduces redox potentials. High-resolution crystal structures, spectroelectrochemical measurements, and high-end computational methods show how this is achieved: each heme iron has a calcium cation positioned next to it at a distance of only 6.

Sheet-on-sheet fixed target data collection devices for serial crystallography at synchrotron and XFEL sources.

Serial crystallography (SX) efficiently distributes over many crystals the radiation dose absorbed during diffraction data acquisition, enabling structure determination of samples at ambient temperature. SX relies on the rapid and reliable replacement of X-ray-exposed crystals with fresh crystals at a rate commensurate with the data acquisition rate. 'Solid supports', also known as 'fixed targets' or 'chips', offer one approach.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore.

Dynamic catcher for stabilization of high-viscosity extrusion jets.

A 'catcher' based on a revolving cylindrical collector is described. The simple and inexpensive device reduces free-jet instabilities inherent to high-viscosity extrusion injection, facilitating delivery of microcrystals for serial diffraction X-ray crystallography.

Structures of an unusual 3-hydroxyacyl dehydratase (FabZ) from a ladderane-producing organism with an unexpected substrate preference.

The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids.

Serial femtosecond crystallography.

With the advent of X-ray Free Electron Lasers (XFELs), new, high-throughput serial crystallography techniques for macromolecular structure determination have emerged. Serial femtosecond crystallography (SFX) and related methods provide possibilities beyond canonical, single-crystal rotation crystallography by mitigating radiation damage and allowing time-resolved studies with unprecedented temporal resolution. This primer aims to assist structural biology groups with little or no experience in serial crystallography planning and carrying out a successful SFX experiment.

Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned.

Upon absorption of a blue-light photon, fatty-acid photodecarboxylase catalyzes the decarboxylation of free fatty acids to form hydrocarbons (for example alkanes or alkenes). The major components of the catalytic mechanism have recently been elucidated by combining static and time-resolved serial femtosecond crystallography (TR-SFX), time-resolved vibrational and electronic spectroscopies, quantum-chemical calculations and site-directed mutagenesis [Sorigué et al. (2021), Science, 372, eabd5687].

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain.

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources.

Crystallographic Studies of Rhodopsins: Structure and Dynamics.

Crystal structures have provided detailed insight in the architecture of rhodopsin photoreceptors. Of particular interest are the protein-chromophore interactions that govern the light-induced retinal isomerization and ultimately induce the large structural changes important for the various biological functions of the family. The reaction intermediates occurring along the rhodopsin photocycle have vastly differing lifetimes, from hundreds of femtoseconds to milliseconds.

Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies.

Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, which hinders structure determination by conventional methods. To automatically extract structural information pertaining to these states, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of methods hitherto not available; (iii) calculates extrapolated structure factors using the various proposed formalisms while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures in real and reciprocal-space. The use of Xtrapol8 could accelerate data processing in kinetic and time-resolved crystallographic studies, and as well foster the identification of drug-targetable states in ligand-based crystallography.

Neodymium as Metal Cofactor for Biological Methanol Oxidation: Structure and Kinetics of an XoxF1-Type Methanol Dehydrogenase.

The methane-oxidizing bacterium Methylacidimicrobium thermophilum AP8 thrives in acidic geothermal ecosystems that are characterized by high degassing of methane (CH), H, HS, and by relatively high lanthanide concentrations. Lanthanides (atomic numbers 57 to 71) are essential in a variety of high-tech devices, including mobile phones. Remarkably, the same elements are actively taken up by methanotrophs/methylotrophs in a range of environments, since their XoxF-type methanol dehydrogenases require lanthanides as a metal cofactor.

Specificity of Small -Type Cytochromes in Anaerobic Ammonium Oxidation.

Anaerobic ammonium oxidation (anammox) is a bacterial process in which ammonium and nitrite are combined into dinitrogen gas and water, yielding energy for the cell. This process relies on a series of redox reactions catalyzed by a set of enzymes, with electrons being shuttled to and from these enzymes, likely by small cytochrome proteins. For this system to work productively, these electron carriers require a degree of specificity toward the various possible redox partners they encounter in the cell.

Dynamics in an unusual acyl carrier protein from a ladderane lipid-synthesizing organism.

Anaerobic ammonium-oxidizing (anammox) bacteria express a distinct acyl carrier protein implicated in the biosynthesis of the highly unusual "ladderane" lipids these organisms produce. This "anammox-specific" ACP, or amxACP, shows several unique features such as a conserved FF motif and an unusual sequence in the functionally important helix III. Investigation of the protein's structure and dynamics, both in the crystal by ensemble refinement and by MD simulations, reveals that helix III adopts a rare six-residue-long 3 -helical conformation that confers a large degree of conformational and positional variability on this part of the protein.

Discerning best practices in XFEL-based biological crystallography - standards for nonstandard experiments.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a novel tool in structural biology. In contrast to conventional crystallography, SFX relies on merging partial intensities acquired with X-ray beams of often randomly fluctuating properties from a very large number of still diffraction images of generally randomly oriented microcrystals. For this reason, and possibly due to limitations of the still evolving data-analysis programs, XFEL-derived SFX data are typically of a lower quality than 'standard' crystallographic data.

Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore.

Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate.

Effect of X-ray free-electron laser-induced shockwaves on haemoglobin microcrystals delivered in a liquid jet.

X-ray free-electron lasers (XFELs) enable obtaining novel insights in structural biology. The recently available MHz repetition rate XFELs allow full data sets to be collected in shorter time and can also decrease sample consumption. However, the microsecond spacing of MHz XFEL pulses raises new challenges, including possible sample damage induced by shock waves that are launched by preceding pulses in the sample-carrying jet.

Structure of the 4-hydroxy-tetrahydrodipicolinate synthase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV and the phylogeny of the aminotransferase pathway.

The enzyme 4-hydroxy-tetrahydrodipicolinate synthase (DapA) is involved in the production of lysine and precursor molecules for peptidoglycan synthesis. In a multistep reaction, DapA converts pyruvate and L-aspartate-4-semialdehyde to 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. In many organisms, lysine binds allosterically to DapA, causing negative feedback, thus making the enzyme an important regulatory component of the pathway.

Structural dynamics in proteins induced by and probed with X-ray free-electron laser pulses.

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.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive.

A nitric oxide-binding heterodimeric cytochrome complex from the anammox bacterium binds to hydrazine synthase.

Anaerobic ammonium oxidation (anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II -type cytochromes called NaxLS, which has distinctive biochemical and spectroscopic properties.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds.