Over the past decade, iterative projection algorithms, an effective approach to recovering phases from a single intensity measurement, have found application in protein crystallography to directly surmount the `phase problem'. However, previous studies have always assumed that some prior knowledge constraints (i.e. a low-resolution envelope about the protein structure in the crystal cell or histogram matching requiring a similar density distribution to the target crystal) must be known for successful phase retrieval, thus hindering its widespread application. In this study, a novel phase-retrieval workflow is proposed that eliminates the need for a reference density distribution by utilizing low-resolution diffraction data in phasing algorithms. The approach involves randomly assigning one out of 12 possible phases at 30° intervals (or two for centric reflections) to produce an initial envelope, which is then refined through density modification after each run of phase retrieval. To evaluate the success of the phase-retrieval procedure, information entropy is introduced as a new metric. This approach was validated using ten protein structures with high solvent content, demonstrating its effectiveness and robustness.

Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of d-mannose and d-glucose, has recently been characterized to have potential for d-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME–d-glucitol and RsME(D254A)–d-glucitol]. RsME possesses the (α/α)6-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loopα7–α8). The RsME–d-glucitol structure showed that loopα7–α8 moves towards d-glucitol and closes the active pocket. Trp251 and Asp254 in loopα7–α8 are only conserved in MEs and interact with d-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)–d-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loopα7–α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.

Aggregates of the antioxidant superoxide dismutase 1 (SOD1) are one of the major contributors to the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in SOD1 lead to an unstable structure and aggregation that perturbs the balance of reactive oxygen species in cells. Oxidation damage to the solvent-exposed Trp32 also causes aggregation of SOD1. Here, the FDA-approved antipsychotic drug paliperidone is identified to interact with Trp32 of SOD1 by structure-based pharmacophore mapping and crystallographic studies. Paliperidone is used for the treatment of schizophrenia. The crystal structure of the complex with SOD1, refined to 2.1 Å resolution, revealed that the ligand binds to the SOD1 β-barrel in the β-strand 2 and 3 regions, which are known to scaffold SOD1 fibrillation. The drug also makes substantial π–π interaction with Trp32. Microscale thermophoresis studies confirm significant binding affinity of the compound, suggesting that the ligand can inhibit or prevent tryptophan oxidation. Thus, the antipsychotic drug paliperidone or a derivative may avert SOD1 aggregation and can be used as a lead for ALS drug development.

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 http://journals.iucr.org/special_issues/2022/RT/.

X-ray crystallography has been invaluable in delivering structural information about proteins. Previously, an approach has been developed that allows high-quality X-ray diffraction data to be obtained from protein crystals at and above room temperature. Here, this previous work is built on and extended by showing that high-quality anomalous signal can be obtained from single protein crystals using diffraction data collected at 220 K up to physiological temperatures. The anomalous signal can be used to directly determine the structure of a protein, i.e. to phase the data, as is routinely performed under cryoconditions. This ability is demonstrated by obtaining diffraction data from model lysozyme, thaumatin and proteinase K crystals, the anomalous signal from which allowed their structures to be solved experimentally at 7.1 keV X-ray energy and at room temperature with relatively low data redundancy. It is also demonstrated that the anomalous signal from diffraction data obtained at 310 K (37°C) can be used to solve the structure of proteinase K and to identify ordered ions. The method provides useful anomalous signal at temperatures down to 220 K, resulting in an extended crystal lifetime and increased data redundancy. Finally, we show that useful anomalous signal can be obtained at room temperature using X-rays of 12 keV energy as typically used for routine data collection, allowing this type of experiment to be carried out at widely accessible synchrotron beamline energies and enabling the simultaneous extraction of high-resolution data and anomalous signal. With the recent emphasis on obtaining conformational ensemble information for proteins, the high resolution of the data allows such ensembles to be built, while the anomalous signal allows the structure to be experimentally solved, ions to be identified, and water molecules and ions to be differentiated. Because bound metal-, phosphorus- and sulfur-containing ions all have anomalous signal, obtaining anomalous signal across temperatures and up to physiological temperatures will provide a more complete description of protein conformational ensembles, function and energetics.

The contrast-variation method in small-angle neutron scattering (SANS) is a uniquely powerful technique for determining the structure of individual components in biomolecular systems containing regions of different neutron scattering length density ρ. By altering the ρ of the target solute and the solvent through judicious incorporation of deuterium, the scattering of desired solute features can be highlighted. Most contrast-variation methods focus on highlighting specific bulk solute elements, but not on how the scattering at specific scattering vectors q, which are associated with specific structural distances, changes with contrast. Indeed, many systems exhibit q-dependent contrast effects. Here, a method is presented for calculating both bulk contrast-match points and q-dependent contrast using 3D models with explicit solute and solvent atoms and SASSENA, an explicit-atom SANS calculator. The method calculates the bulk contrast-match points within 2.4% solvent D2O accuracy for test protein–nucleic acid and lipid nanodisc systems. The method incorporates a general model for the incorporation of deuterium at non-exchangeable sites that was derived by performing mass spectrometry on green fluorescent protein. The method also decomposes the scattering profile into its component parts and identifies structural features that change with contrast. The method is readily applicable to a variety of systems, will expand the understanding of q-dependent contrast matching and will aid in the optimization of next-generation neutron scattering experiments.

Alexei Vagin is remembered.

A figure in the article by Barbarin-Bocahu & Graille [(2022), Acta Cryst. D78, 517–531] is corrected.

l-Proline hydroxylase is a member of the non-heme Fe2+/α-ketoglutarate (AKG)-dependent hydroxylase family that catalyzes the reaction from l-proline to hydroxy-l-proline, which is widely used in drug synthesis, biochemistry, food supplementation and cosmetic industries. Here, the first crystal structure of l-proline trans-hydroxylase and its complexes with substrate and product are reported, which reveal the structural basis of trans–cis proline hydroxylation selectivity. Structure comparison with other AKG-dependent hydroxylases identifies conserved amino acid residues, which may serve as signatures of in-line or off-line AKG binding modes in the AKG-dependent enzyme family.