Current trends in macromolecular model refinement and validation.

The Guest Editors provide an introduction to the special issue of articles based on talks at the CCP4 Study Weekend 2022, which is available at https://journals.iucr.org/special_issues/2023/CCP42022/.

Accelerating the discovery of new materials with deep learning.

The use of deep learning and ideas from image recognition to accelerate data analysis and hence discovery of novel materials is described. A particular focus is given to the field of energy research.

Structure and activation mechanism of the human liver-type glutaminase GLS2.

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.

The predictive power of data-processing statistics.

This study describes a method to estimate the likelihood of success in determining a macromolecular structure by X-ray crystallography and experimental single-wavelength anomalous dispersion (SAD) or multiple-wavelength anomalous dispersion (MAD) phasing based on initial data-processing statistics and sample crystal properties. Such a predictive tool can rapidly assess the usefulness of data and guide the collection of an optimal data set. The increase in data rates from modern macromolecular crystallography beamlines, together with a demand from users for real-time feedback, has led to pressure on computational resources and a need for smarter data handling. Statistical and machine-learning methods have been applied to construct a classifier that displays 95% accuracy for training and testing data sets compiled from 440 solved structures. Applying this classifier to new data achieved 79% accuracy. These scores already provide clear guidance as to the effective use of computing resources and offer a starting point for a personalized data-collection assistant.

How best to use photons.

Strategies for collecting X-ray diffraction data have evolved alongside beamline hardware and detector developments. The traditional approaches for diffraction data collection have emphasised collecting data from noisy integrating detectors (i.e. film, image plates and CCD detectors). With fast pixel array detectors on stable beamlines, the limiting factor becomes the sample lifetime, and the question becomes one of how to expend the photons that your sample can diffract, i.e. as a smaller number of stronger measurements or a larger number of weaker data. This parameter space is explored via experiment and synthetic data treatment and advice is derived on how best to use the equipment on a modern beamline. Suggestions are also made on how to acquire data in a conservative manner if very little is known about the sample lifetime.

DIALS: implementation and evaluation of a new integration package.

The DIALS project is a collaboration between Diamond Light Source, Lawrence Berkeley National Laboratory and CCP4 to develop a new software suite for the analysis of crystallographic X-ray diffraction data, initially encompassing spot finding, indexing, refinement and integration. The design, core algorithms and structure of the software are introduced, alongside results from the analysis of data from biological and chemical crystallography experiments.

Background modelling of diffraction data in the presence of ice rings.

An algorithm for modelling the background for each Bragg reflection in a series of X-ray diffraction images containing Debye-Scherrer diffraction from ice in the sample is presented. The method involves the use of a global background model which is generated from the complete X-ray diffraction data set. Fitting of this model to the background pixels is then performed for each reflection independently. The algorithm uses a static background model that does not vary over the course of the scan. The greatest improvement can be expected for data where ice rings are present throughout the data set and the local background shape at the size of a spot on the detector does not exhibit large time-dependent variation. However, the algorithm has been applied to data sets whose background showed large pixel variations (variance/mean > 2) and has been shown to improve the results of processing for these data sets. It is shown that the use of a simple flat-background model as in traditional integration programs causes systematic bias in the background determination at ice-ring resolutions, resulting in an overestimation of reflection intensities at the peaks of the ice rings and an underestimation of reflection intensities either side of the ice ring. The new global background-model algorithm presented here corrects for this bias, resulting in a noticeable improvement in R factors following refinement.

AUSPEX: a graphical tool for X-ray diffraction data analysis.

In this paper, AUSPEX, a new software tool for experimental X-ray data analysis, is presented. Exploring the behaviour of diffraction intensities and the associated estimated uncertainties facilitates the discovery of underlying problems and can help users to improve their data acquisition and processing in order to obtain better structural models. The program enables users to inspect the distribution of observed intensities (or amplitudes) against resolution as well as the associated estimated uncertainties (sigmas). It is demonstrated how AUSPEX can be used to visually and automatically detect ice-ring artefacts in integrated X-ray diffraction data. Such artefacts can hamper structure determination, but may be difficult to identify from the raw diffraction images produced by modern pixel detectors. The analysis suggests that a significant portion of the data sets deposited in the PDB contain ice-ring artefacts. Furthermore, it is demonstrated how other problems in experimental X-ray data caused, for example, by scaling and data-conversion procedures can be detected by AUSPEX.

Inter-domain communication of human cystathionine ß-synthase: structural basis of S-adenosyl-L-methionine activation.

Cystathionine ß-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe(443), Asp(444), Gln(445), and Asp(538)) and for AdoMet-driven inter-domain communication (Phe(443), Asp(538)). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.

Ribosomal oxygenases are structurally conserved from prokaryotes to humans.

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

Human UTY(KDM6C) is a male-specific Nϵ-methyl lysyl demethylase.

The Jumonji C lysine demethylases (KDMs) are 2-oxoglutarate- and Fe(II)-dependent oxygenases. KDM6A (UTX) and KDM6B (JMJD3) are KDM6 subfamily members that catalyze demethylation of N(ϵ)-methylated histone 3 lysine 27 (H3K27), a mark important for transcriptional repression. Despite reports stating that UTY(KDM6C) is inactive as a KDM, we demonstrate by biochemical studies, employing MS and NMR, that UTY(KDM6C) is an active KDM. Crystallographic analyses reveal that the UTY(KDM6C) active site is highly conserved with those of KDM6B and KDM6A. UTY(KDM6C) catalyzes demethylation of H3K27 peptides in vitro, analogously to KDM6B and KDM6A, but with reduced activity, due to point substitutions involved in substrate binding. The results expand the set of human KDMs and will be of use in developing selective KDM inhibitors.

Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations.

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming.

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)(18)-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K(528)R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Identification of macrodomain proteins as novel O-acetyl-ADP-ribose deacetylases.

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD(+) to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD(+)-dependent deacetylation.

Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.

The structure of the membrane integral rotor ring of the proton translocating F(1)F(0) ATP synthase from spinach chloroplasts was determined to 3.8 A resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c(11) rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6-10.8 A apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu(61) in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu(61) is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu(61) by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.