The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

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The analysis of large structural databases reveals general features and relationships among proteins, providing useful insight. A different approach is required to characterize ubiquitous secondary‐structure elements, where flexibility is essential in order to capture small local differences. The ALEPH software is optimized for the analysis and the extraction of small protein folds by relying on their geometry rather than on their sequence. The annotation of the structural variability of a given fold provides valuable information for fragment‐based molecular‐replacement methods, in which testing alternative model hypotheses can succeed in solving difficult structures when no homology models are available or are successful. ARCIMBOLDO_BORGES combines the use of composite secondary‐structure elements as a search model with density modification and tracing to reveal the rest of the structure when both steps are successful. This phasing method relies on general fold libraries describing variations around a given pattern of β‐sheets and helices extracted using ALEPH. The program introduces characteristic vectors defined from the main‐chain atoms as a way to describe the geometrical properties of the structure. ALEPH encodes structural properties in a graph network, the exploration of which allows secondary‐structure annotation, decomposition of a structure into small compact folds, generation of libraries of models representing a variation of a given fold and finally superposition of these folds onto a target structure. These functions are available through a graphical interface designed to interactively show the results of structure manipulation, annotation, fold decomposition, clustering and library generation. ALEPH can produce pictures of the graphs, structures and folds for publication purposes.

UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria. IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite’s ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

Structure predictions have matched the accuracy of experimental structures from close homologues, providing suitable models for molecular replacement phasing. Even in predictions that present large differences due to the relative movement of domains or poorly predicted areas, very accurate regions tend to be present. These are suitable for successful fragment-based phasing as implemented in ARCIMBOLDO. The particularities of predicted models are inherently addressed in the new predicted_model mode, rendering preliminary treatment superfluous but also harmless. B-value conversion from predicted LDDT or error estimates, the removal of unstructured polypeptide, hierarchical decomposition of structural units from domains to local folds and systematically probing the model against the experimental data will ensure the optimal use of the model in phasing. Concomitantly, the exhaustive use of models and stereochemistry in phasing, refinement and validation raises the concern of crystallographic model bias and the need to critically establish the information contributed by the experiment. Therefore, in its predicted_model mode ARCIMBOLDO_SHREDDER will first determine whether the input model already constitutes a solution or provides a straightforward solution with Phaser. If not, extracted fragments will be located. If the landscape of solutions reveals numerous, clearly discriminated and consistent probes or if the input model already constitutes a solution, model-free verification will be activated. Expansions with SHELXE will omit the partial solution seeding phases and all traces outside their respective masks will be combined in ALIXE, as far as consistent. This procedure completely eliminates the molecular replacement search model in favour of the inferences derived from this model. In the case of fragments, an incorrect starting hypothesis impedes expansion. The predicted_model mode has been tested in different scenarios.

Fragment‐based molecular‐replacement methods can solve a macromolecular structure quasi‐ab initio. ARCIMBOLDO, using a common secondary‐structure or tertiary‐structure template or a library of folds, locates these with Phaser and reveals the rest of the structure by density modification and autotracing in SHELXE. The latter stage is challenging when dealing with diffraction data at lower resolution, low solvent content, high β‐sheet composition or situations in which the initial fragments represent a low fraction of the total scattering or where their accuracy is low. SEQUENCE SLIDER aims to overcome these complications by extending the initial polyalanine fragment with side chains in a multisolution framework. Its use is illustrated on test cases and previously unknown structures. The selection and order of fragments to be extended follows the decrease in log‐likelihood gain (LLG) calculated with Phaser upon the omission of each single fragment. When the starting substructure is derived from a remote homolog, sequence assignment to fragments is restricted by the original alignment. Otherwise, the secondary‐structure prediction is matched to that found in fragments and traces. Sequence hypotheses are trialled in a brute‐force approach through side‐chain building and refinement. Scoring the refined models through their LLG in Phaser may allow discrimination of the correct sequence or filter the best partial structures for further density modification and autotracing. The default limits for the number of models to pursue are hardware dependent. In its most economic implementation, suitable for a single laptop, the main‐chain trace is extended as polyserine rather than trialling models with different sequence assignments, which requires a grid or multicore machine. SEQUENCE SLIDER has been instrumental in solving two novel structures: that of MltC from 2.7 Å resolution data and that of a pneumococcal lipoprotein with 638 residues and 35% solvent content.

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.

Structural predictions have matched the accuracy of experimental structures in the case of close homologues, outperformed docking methods for multimeric complexes and helped sampling the conformational landscape of transporters and receptors. Such successes prompt the question whether predictions can be used to relate experimental structures in the context of available knowledge. LysR-type transcriptional regulators (LTTR) constitute the most common family of bacterial regulators. Intriguingly, their experimental structures are remarkably diverse. The active species, composed of flexible monomers dimerizing through their N- and C-terminal domains in a circular arrangement, differ across LTTR, due to intrinsic sequence differences or because crystals stabilize diverse snapshots of a common dynamic mechanism. We have used AlphaFold2 (AF) to interrogate the experimental AtzR structure in the context of predictions guided towards the different hetero-multimeric conformations known for other LTTR. Our approach drives AF prediction with the structure-based selection of the information input through sequence alignment and template conformation, linked to examination of the energy with PISA and interactions with ALEPH.