Room-temperature biological crystallography has seen a resergence in recent years and a collection of articles recently published in IUCrJ, Acta Cryst. D Structural Biology and Acta Cryst. F Structural Biology Communications, have been collected together to produce a virtual special issue at https://journals.iucr.org/special_issues/2022/RT/.

The determination of the structure of the multifunctional globin dehaloperoxidase using multiple room-temperature methods is described. Structures obtained by serial femtosecond crystallography, serial synchrotron crystallography, neutron diffraction and serial Laue crystallography are compared and two oxidation states of the enzyme are contrasted.

Serial crystallography relies upon the growth of microcrystals at high concentration. An LCP crystallization protocol for the Blastochloris viridis photosynthetic reaction centre that utilizes seeding from detergent-grown crystals is reported.

Analysis of high-resolution data indicated that X-ray damage has little impact on conformational ensemble information from protein crystals at room temperature (277 K). In contrast, X-ray damage can alter conformational distributions obtained from cryo-cooled crystals under common experimental conditions, complicating structural interpretations in some instances. The results presented provide a strong motivation for the expanded use of room-temperature X-ray crystallography.

A room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed and applied to study ligand binding and protein plasticity.

X-ray crystallography at variable tem­per­ature for SARS-CoV-2 M^pro reveals a com­plex conformational landscape, including a mobile metal at the catalytic dyad, mercurial conformational heterogeneity at various sites, and an intra­molecular network bridging the active site and dimer inter­face.

The use of room-temperature crystallography to probe protein dynamics on the second-to-minute timescale is demonstrated by monitoring the release of a photoequilibrium induced in crystals of a LOV domain. The slow protein dynamics components include the relaxation of a photoadduct and a crystalline phase transition. The result of the latter is the formation of a non-crystallographic dimer in which the C-termini of the two monomers reorder in distinct conformations on different timescales.

The structure of the full-length Drosophila cryptochrome as solved by fixed-target serial synchrotron crystallography at the Cornell High Energy Synchrotron Source is presented. Scaling and filtering methods were performed to improve the overall quality of the final model.

Room-temperature crystallography enables researchers to resolve the conformational heterogeneity of structures. Here, the native SAD phasing of four structures at 295 K highlights the strengths of room-temperature diffraction experiments, including detailed anomalous difference maps and alternate conformations that are well supported by the electron density.

Using the CFEL TapeDrive, minimization of sample consumption and data collection times needed for serial synchrotron crystallography (SSX) were achieved and the first room-temperature structures of two enzymes are described as well as an assessment of the benefit of SSX for a radiation-sensitive protein.

An in situ X-ray diffraction measurement system using a crystallization plate has been constructed at the SPring-8 protein crystallography beamline. Utilizing small-wedge measurements and incorporating a liquid dispenser to prepare protein–ligand complex crystals, this system will make ligand screening possible.

The first serial synchrotron crystallographic structure of apo PTP1B is reported and the conformational heterogeneity at several allosteric sites is compared with that seen in prior structures.

Key physical principles and methods for collecting crystallographic data from biomolecular systems at room temperature and, more generally, at temperatures between ∼200 and ∼350 K are reviewed and discussed.

Anomalous information was obtained from model lysozyme, thaumatin and proteinase K crystals from 220 K up to physiological temperatures.

Serial femtosecond X-ray crystallography was performed using a liquid-jet system on bacterial copper amine oxidase microcrystals anaerobically premixed with an amine substrate. The structure determined at 1.94 Å resolution revealed catalytic intermediates in two reduced forms, confirming that anaerobic conditions were well maintained throughout the measurements.