A short story of the long road to cryo-EM in Portugal

This letter describes the development of cryo-EM in Portugal, highlighting a strategy based on research training and the establishment of a national facility with regional support nodes. Our experience demonstrates how access to European research infrastructures can enable the creation of sustainable national platforms without investment in high-end microscopes.

Cryo-EM reveals ArnA contamination during purification of a ciliary protein complex

ArnA contamination was independently observed during the purification of a soluble, low-yield protein complex, leading to an unintended 3.23 Å resolution cryo-EM structure. This work broadens recent findings and highlights ArnA as a recurrent His-tag-associated contaminant in Escherichia coli purification workflows.

A cryo-EM processing pipeline for microtubules using CryoSPARC

Cryo-EM reconstructions of microtubules are currently technically challenging and time-consuming for average users. A cryo-EM processing pipeline for microtubules using CryoSPARC has been developed that overcomes many of these current issues.

Alphafuser: a parsimonious approach to predicting higher-order protein complexes

Alphafuser is a structure-prediction pipeline that integrates experimental interaction data with AlphaFold-based modeling to systematically assemble multiprotein complexes in a computationally efficient manner. By implementing an ipTM-based pruning algorithm and validating against known structures and experimental assays, Alphafuser enables the accurate identification of higher-order assemblies from large interactome datasets.

Generation and modulation of catalytically relevant states of a dye-decolourizing peroxidase using time-resolved serial femtosecond crystallography with drop-on-chip mixing and X-ray-driven reduction

We characterize the formation of a ferryl state of a dye-decolourizing peroxidase by drop-on-chip serial femtosecond crystallography and also the modulation of the heme redox state using dose-resolved serial synchrotron crystallography.

Electrons and X-rays for diffraction and imaging

The use of X-rays and electrons for diffraction and imaging of soft biological samples is compared.

Structures of variants of Escherichia coli flavodiiron-type nitric oxide reductase reveal changes in the di-iron site

Flavodiiron proteins (FDPs) are di-iron enzymes that reduce NO and/or O₂ with distinct substrate selectivity. We have produced E. coli FDP variants targeting second coordination-sphere residues and determined their structures. Although the kinetics remained unchanged, the E. coli FDP S262Y variant showed X-ray radiation-induced photoreduction, suggesting increased structural sensitivity without altered selectivity.

Damage-limited resolution for X-ray and electron microscopy of organic specimens

Analytical expressions for the damage-limited resolution are developed and applied to X-ray and electron imaging of beam-sensitive specimens. The findings will guide future microscopy and instrument design.

Radiation damage in sub-Ångström resolution macromolecular crystallography: a low-dose study

This first systematic radiation-damage study at 0.55 Å resolution reveals extensive small conformational changes in response to dose. The findings demonstrate the benefits of using large crystals fully bathed in a large `top-hat' beam with a uniform fluence profile, together with low-dose data-collection protocols, for sub-Ångström structure investigations on macromolecules.

Radiation-induced peroxide rupture and its temperature-dependent repair probed by homogeneous X-ray irradiation

This manuscript investigates the X-ray-induced radiolysis of the catalytic C5-peroxide adduct in urate oxidase crystals, utilizing a top-hat X-ray beam to monitor peroxide occupancy across extensive dose series at both 100 K and room temperature. The study reveals a kinetic phase transition where the peroxide decays rapidly via zero-order kinetics at room temperature, whereas at 100 K the decay is retarded and follows first-order kinetics due to an efficient recombination mechanism involving trapped oxygen.

Mapping the effects of specific radiation damage and solvent radiolysis in buffers and crystals with online UV–Vis absorption spectroscopy

Radiation-induced changes during X-ray crystallographic data collection can compromise concurrent spectroscopic studies. Suitable protocols to detect and identify spectral artefacts from crystallization buffers and cryoprotectants are essential for the reliable analysis of chromophoric ligands in macromolecules.

Optimal 1TEL–target protein linker character is target protein-dependent

In this study, we examine the effect of short to medium-length flexible, semi-flexible and rigid linkers on the crystallization of a DARPin or the TNK1 UBA domain fused to the 1TEL protein crystallization chaperone, demonstrating that while rigid linkers can impair crystallization and reduce diffraction quality, the ideal linker character remains target protein-dependent.

Model-dependent and model-independent visualization of hydrogen atoms in high-resolution cryoEM maps

A hidden hydrogen peak as a shoulder feature of a larger carbon peak can be computationally decomposed in a model-independent visualization method by calculating the one-dimensional profile difference between the right side of the profile that adjoins this H atom and the left side that does not.

Gaining insights into MmpL3: combining structural and computational approaches to unlock transport and inhibitor-binding mechanisms

This study investigates the mycobacterial membrane protein MmpL3, a key target for new antituberculosis drugs, by combining high-resolution crystallography and computational simulations. It reveals how the inhibitor UPAR-1109 binds to MmpL3, providing detailed insights into the function and flexibility of the protein and supporting the rational design of next-generation antimycobacterial therapies.

Wolfram Saenger (1939–2026)

Wolfram Saenger is remembered.