Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway, and the mode of action of a RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the bacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear nonheme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are rare examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide allowing them to be exported by the ubiquitous Sec pathway. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by complexing excess metal ions. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in bacteria.

Femtosecond transient soft X-ray absorption spectroscopy (XAS) is a very promising technique that can be employed at X-ray free-electron lasers (FELs) to investigate out-of-equilibrium dynamics for material and energy research. Here, a dedicated setup for soft X-rays available at the Spectroscopy and Coherent Scattering (SCS) instrument at the European X-ray Free-Electron Laser (European XFEL) is presented. It consists of a beam-splitting off-axis zone plate (BOZ) used in transmission to create three copies of the incoming beam, which are used to measure the transmitted intensity through the excited and unexcited sample, as well as to monitor the incoming intensity. Since these three intensity signals are detected shot by shot and simultaneously, this setup allows normalized shot-by-shot analysis of the transmission. For photon detection, an imaging detector capable of recording up to 800 images at 4.5 MHz frame rate during the FEL burst is employed, and allows a photon-shot-noise-limited sensitivity to be approached. The setup and its capabilities are reviewed as well as the online and offline analysis tools provided to users.

We present in this work a first X‐ray Absorption Spectroscopy study of the interactions of Zn with human BST2/tetherin and SARS‐CoV‐2 orf7a proteins as well as with some of their complexes. The analysis of the XANES region of the measured spectra shows that Zn binds to BST2, as well as to orf7a, thus resulting in the formation of BST2‐orf7a complexes. This structural information confirms the the conjecture, recently put forward by some of the present Authors, according to which the accessory orf7a (and possibly also orf8) viral protein are capable of interfering with the BST2 antiviral activity. Our explanation for this behavior is that, when BST2 gets in contact with Zn bound to the orf7a Cys₁₅ ligand, it has the ability of displacing the metal owing to the creation of a new disulfide bridge across the two proteins. The formation of this BST2‐orf7a complex destabilizes BST2 dimerization, thus impairing the antiviral activity of the latter.

A proposal for building a Free Electron Laser, EuPRAXIA@SPARC_LAB, at the Laboratori Nazionali di Frascati, is at present under consideration. This FEL facility will provide a unique combination of a high brightness GeV-range electron beam generated in a X-band RF linac, a 0.5 PW-class laser system and the first FEL source driven by a plasma accelerator. The FEL will produce ultra-bright pulses, with up to 10 ${}^{12}$ photons/pulse, femtosecond timescale and wavelength down to 3 nm, which lies in the so called “water window”. The experimental activity will be focused on the realization of a plasma driven short wavelength FEL able to provide high-quality photons for a user beamline. In this paper, we describe the main classes of experiments that will be performed at the facility, including coherent diffraction imaging, soft X-ray absorption spectroscopy, Raman spectroscopy, Resonant Inelastic X-ray Scattering and photofragmentation measurements. These techniques will allow studying a variety of samples, both biological and inorganic, providing information about their structure and dynamical behavior. In this context, the possibility of inducing changes in samples via pump pulses leading to the stimulation of chemical reactions or the generation of coherent excitations would tremendously benefit from pulses in the soft X-ray region. High power synchronized optical lasers and a TeraHertz radiation source will indeed be made available for THz and pump–probe experiments and a split-and-delay station will allow performing XUV-XUV pump–probe experiments.

The redox activity of Cu ions bound to the amyloid‐β (Aβ) peptide is implicated as a source of oxidative stress in the context of Alzheimer's disease. In order to explain the efficient redox cycling between Cu^II‐Aβ (distorted square‐pyramidal) and Cu^I‐Aβ (digonal) resting states, the existence of a low‐populated “in‐between” state, prone to bind Cu in both oxidation states, has been postulated. Here, we exploited the partial X‐ray induced photoreduction at 10 K, followed by a thermal relaxation at 200 K, to trap and characterize by X‐ray Absorption Spectroscopy (XAS) a partially reduced Cu‐Aβ_1–16 species different from the resting states. Remarkably, the XAS spectrum is well‐fitted by a previously proposed model of the “in‐between” state, hence providing the first direct spectroscopic characterization of an intermediate state. The present approach could be used to explore and identify the catalytic intermediates of other relevant metal complexes.

In this paper we prove in the exemplary case of the amyloid-β peptide in complex with Cu(ii) that at the current low temperatures employed in XAS experiments, the time needed for collecting a good quality XAS spectrum is significantly shorter than the time after which structural damage becomes appreciable. Our method takes advantage of the well-known circumstance that the transition of Cu from the oxidized to the reduced form under ionizing radiation can be quantified by monitoring a characteristic peak in the pre-edge region. We show that there exists a sufficiently large time window in which good XAS spectra can be acquired before the structure around the oxidized Cu(ii) ion reorganizes itself into the reduced Cu(i) “resting” structure. We suggest that similar considerations apply to other cases of biological interest, especially when dealing with macromolecules in complex with transition metal ions.