Molecular and structural basis of oligopeptide recognition by the Ami transporter system in pneumococci.

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.

Predicted models and CCP4.

In late 2020, the results of CASP14, the 14th event in a series of competitions to assess the latest developments in computational protein structure-prediction methodology, revealed the giant leap forward that had been made by Google's Deepmind in tackling the prediction problem. The level of accuracy in their predictions was the first instance of a competitor achieving a global distance test score of better than 90 across all categories of difficulty. This achievement represents both a challenge and an opportunity for the field of experimental structural biology. For structure determination by macromolecular X-ray crystallography, access to highly accurate structure predictions is of great benefit, particularly when it comes to solving the phase problem. Here, details of new utilities and enhanced applications in the CCP4 suite, designed to allow users to exploit predicted models in determining macromolecular structures from X-ray diffraction data, are presented. The focus is mainly on applications that can be used to solve the phase problem through molecular replacement.

The CCP4 suite: integrative software for macromolecular crystallography.

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.

Verification: model-free phasing with enhanced predicted models in ARCIMBOLDO_SHREDDER.

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.

SPACA6 ectodomain structure reveals a conserved superfamily of gamete fusion-associated proteins.

SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of the SPACA6 ectodomain at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like ß-sandwich connected via a quasi-flexible linker. This structure is reminiscent of IZUMO1, another gamete fusion-associated protein, making SPACA6 and IZUMO1 founding members of a superfamily of fertilization-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion.

ALIXE: a phase-combination tool for fragment-based molecular replacement.

Fragment-based molecular replacement exploits the use of very accurate yet incomplete search models. In the case of the ARCIMBOLDO programs, consistent phase sets produced from the placement and refinement of fragments with Phaser can be combined in order to increase their signal before proceeding to the step of density modification and autotracing with SHELXE. The program ALIXE compares multiple phase sets, evaluating mean phase differences to determine their common origin, and subsequently produces sets of combined phases that group consistent solutions. In this work, its use on different scenarios of very partial molecular-replacement solutions and its performance after the development of a much-optimized set of algorithms are described. The program is available both standalone and integrated within the ARCIMBOLDO programs. ALIXE has been analysed to identify its rate-limiting steps while exploring the best parameterization to improve its performance and make this software efficient enough to work on modest hardware. The algorithm has been parallelized and redesigned to meet the typical landscape of solutions. Analysis of pairwise correlation between the phase sets has also been explored to test whether this would provide additional insight. ALIXE can be used to exhaustively analyse all partial solutions produced or to complement those already selected for expansion, and also to reduce the number of redundant solutions, which is particularly relevant to the case of coiled coils, or to combine partial solutions from different programs. In each case parallelization and optimization to provide speedup makes its use amenable to typical hardware found in crystallography. ARCIMBOLDO_BORGES and ARCIMBOLDO_SHREDDER now call on ALIXE by default.

Clinical impact of the ERBP Working Group 2010 Recommendations for the anemia management in chronic kidney disease not on dialysis: ACERCA study.

BACKGROUND AND OBJECTIVE: The Anemia Working Group of ERBP in 2010 recommended a target hemoglobin (Hb) level in the range of 11-12 g/dL, without intentionally exceeding 13 g/dL during the treatment with erythropoiesis stimulating agents (ESAs). This study evaluated if there was a clinical impact of this statement in the anemia management of chronic kidney disease (CKD) patients treated with ESAs not on dialysis in routine clinical practice in Spain. METHODS: This was an observational and cross-sectional study carried out in CKD patients not on dialysis in Spain who initiated ESA treatment (naïve), or were shifted from a previous ESA to another ESAs (converted) since January 2011. RESULTS: Of 441 patients evaluated, 67.6% were naïve and 32.4% were converted. At the study visit, 42.5% of naïve patients achieved the Hb target of 11-12 g/dL, with a mean Hb of 11.3±1.3 g/dL (vs 10.1±0.9 g/dL at the start of ESA therapy). Only 35.3% of converted patients maintained Hb levels within the recommended target at the study visit. Yet, 8.2% of naïve patients and 7.9% of those converted had Hb levels >13 g/dL. Hb levels were similar across subgroups of patients, regardless of the presence of significant comorbidities. CONCLUSIONS: Anemia management in CKD patients treated with ESAs by Spanish nephrologists seems to be aimed at preventing Hb levels <11 g/dL, while <50% of patients were within the narrow recommended Hb target range. This, together with the lack of individualization in Hb targets according to patients' comorbidities show that there is still room for improvement in renal anemia management in the clinical setting.