Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments

It is shown that the anisotropy of anomalous scattering (AAS) is a significant and ubiquitous effect in data sets collected at an absorption edge and that its exploitation can substantially enhance the phasing power of single- or multi-wavelength anomalous diffraction. The improvements in the phases are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment

Polarization-dependence of anomalous scattering in brominated DNA and RNA molecules, and importance of crystal orientation in single- and multiple-wavelength anomalous diffraction phasing

In this paper the anisotropy of anomalous scattering at the Br K-absorption edge in brominated nucleotides is investigated, and it is shown that this effect can give rise to a marked directional dependence of the anomalous signal strength in X-ray diffraction data. This implies that choosing the correct orientation for crystals of such molecules can be a crucial determinant of success or failure when using single- and multiple-wavelength anomalous diffraction (SAD or MAD) methods to solve their structure. In particular, polarized absorption spectra on an oriented crystal of a brominated DNA molecule were measured, and were used to determine the orientation that yields a maximum anomalous signal in the diffraction data. Out of several SAD data sets, only those collected at or near that optimal orientation allowed interpretable electron density maps to be obtained. The findings of this study have implications for instrumental choices in experimental stations at synchrotron beamlines, as well as for the development of data collection strategy programs

Plasma Membrane Dynamics Regulating the PD-1/PD-L1 Pathway

The PD-1/PD-L1 pathway in T lymphocytes is emerging as one of the most promising targets for cancer immunotherapy but few details are yet known about how its inhibitory influence is achieved. This work set out to investigate the role of plasma membrane dynamics, on both T cells and tumour cells, in modulating the pathway. In eukaryotic cells, calcium influx leads to rapid changes in the plasma membrane. Exocytosis, phospholipid scrambling, membrane shedding and massive endocytosis can all occur. The calcium sensors for these processes, their inter-dependence and their effects on transmembrane protein expression remain largely unknown. Here we show that the ion channel TMEM16F is the calcium sensor for large exocytosis and that phospholipid scrambling and microvesicle shedding are coupled to exocytosis. The absence of TMEM16F not only abrogates exocytosis but also results in massive endocytosis in response to calcium. Intracellular polyamines regulate these phenotypes, switching cell responses from massive exo- to endocytosis by blocking the TMEM16F conductance. This massive endocytosis also occurs during apoptosis via a calcium-independent mechanism. In lymphocytes, PD-1 participates selectively in both shedding and massive endocytosis, targeting that depends on the PD-1 transmembrane region, independent of actin and classical protein adaptors. We also found that PD-L1 on tumour cells can be transferred to lymphocytes, and be maintained in stable complex with PD-1 on in vitro and in vivo. This may modulate the PD-L1/PD-1 pathway in tumours. Together, these results provide new insights into the plasma membrane reorganization that occurs following calcium transients, establishes a mechanism of PD-1 regulation dependent solely on protein-membrane interaction, and also introduces a new modality by which PD-L1:PD-1 interactions can be sustained beyond cell-cell contact in tumours

Re-refinement from deposited X-ray data can deliver improved models for most PDB entries

An evaluation of validation and real-space intervention possibilities for improving existing automated (re-)refinement methods

Méthode agile pour la conception collaborative multidisciplinaire de systèmes intégrés : application à la mécatronique

This work focuses on the multidisciplinary and collaborative design of integrated systems. These systems are subject to an ever increasing number of requirements, leading to the need for more comprehensive functional and spatial integration. These different types of product integration are also at the origin of organizational complexity. This complexity arises not only from the great number of actors performing various engineering activities but also from the diversity of disciplines involved (designated in this manuscript as “multidisciplinary integration”). To encourage this multidisciplinary integration, “preliminary design” and “detailed design” have been identified as the most significant steps, especially since they are characterized by the collaboration of multiple experts handling a large number of product definition’ technical data. Systems that have been designed thanks to multidisciplinary approaches are generally poorly integrated. This is partially due to the compartmentalization of disciplines, as well as to the “project-planned” method, where project planning is predominant and information is mainly spread out “top-down”. To ensure better cooperation between the various disciplines, to enable decision making based on operational indicators and to analyze and understand the multidisciplinary integration processes, a method inspired by the founding principles of agile methods (the agile manifesto) is proposed for the collaborative design of integrated systems. This work is based on three complementary concepts. The first is, the Collaborative Actions Framework, an operational framework for collaboration around actions. One objective of this framework is to improve the collaboration among designers, whatever their disciplinary origin. It also ensures traceability between decision making and corrections/changes made to technical data. This traceability is made possible by the useof the second concept, called Workspace. Even if this term is already well known, we propose a new definition/usage to transform it into collaboration spaces. This concept offers great possibilities, including the continuous delivering/sharing of experts’ contributions, multidisciplinary integration and change validation. The exchange of technical data between workspaces, or simultaneous work on the same data, relies on the ability to manage several parallel versions of the same item into a single datamanagement system. These opportunities are offered by the third concept, called Branch & Merge. Finally, these three concepts are illustrated through a scenario and a computer prototype. A mechatronic product, “the synergistic combination of mechanical and electrical engineering, computer science, and information technology” (Harashima et al., 1996), is used to illustrate the opportunities offered by our work in terms of multidisciplinary integration during collaborative design.Ces travaux portent sur la conception multidisciplinaire de systèmes intégrés. Ces systèmes sont soumis à un nombre d’exigences toujours croissant, entraînant des besoins en termes d’intégration fonctionnelle et spatiale. Ces différents types d’intégration relative au produit sont également la source d’une complexité organisationnelle, provenant à la fois de la multitude d’acteurs réalisant différentes activités d’ingénierie, mais également de la diversité des domaines impliqués, désignée dans ce manuscrit par « intégration multidisciplinaire ». Pour favoriser cette intégration multidisciplinaire, les phases de « conception préliminaire » et de « conception détaillée » ont été identifiées comme déterminantes, notamment car elles se caractérisent par la collaboration de nombreux experts, manipulant un grand nombre de données techniques de définition. Les systèmes conçus lors de conceptions multidisciplinaires restent faiblement intégrés. Cela est en partie dû au cloisonnement entre les disciplines et à un mode d’organisation projet basé sur une planification prédominante, caractérisé notamment par une diffusion de l’information principalement descendante (top-down). Afin d’assurer une meilleure collaboration entre ces différentes disciplines, de permettre des prises de décision éclairées par des indicateurs opérationnels et de pouvoir analyser et mieux comprendre les phénomènes d’intégration des expertises, l’introduction d’une méthode inspirée des principes fondateurs des méthodes agiles est proposée pour la conception collaborative de systèmes intégrés.La contribution de ces travaux s’appuie sur trois concepts complémentaires. Le premier, intitulé Collaborative Actions Framework correspond à un cadre de collaboration opérationnelle autour d’actions. Un des objectifs de ce framework est de faciliter la collaboration des acteurs des projets de conception, quelle que soit leur origine disciplinaire, mais également d’assurer une traçabilité entre les prises de décision et les corrections/modifications apportées sur les données techniques. Cette traçabilité est rendue possible grâce aux liens existants avec le second concept intitulé Workspace. Apportant un nouvel éclairage sur les possibilités offertes par la collaboration autour de ces espaces de collaboration, ce concept offre un certain nombre de possibilités,notamment la mise en commun continue des travaux, l’intégration multidisciplinaire et la validation des modifications. Les échanges de données techniques entre les workspaces, ou le travail simultané sur les mêmes données techniques, s’appuient quant à eux sur la possibilité de pouvoir gérer de façon parallèle différentes versions d’une même donnée technique. Ces possibilités sont proposées par le troisième concept, intitulé branch & merge, qui permet également à différents acteurs de travailler simultanément sur les mêmes données. Enfin, ces trois concepts sont ensuite illustrés par l’intermédiaire d’un démonstrateur composé d’un scénario et d’un prototype informatique. Un produit mécatronique, combinaison synergique et systémique de la mécanique, de l'électronique et de l'informatique temps réel, est utilisé afin d’illustrer les possibilités offertes par nos travaux en termes d'intégration multidisciplinaire lors de la conception collaborative

Instrument-independent specification of the diffraction geometry and polarization state of the incident X-ray beam

This work augments the proposal of Schwarzenbach & Flack [J. Appl. Cryst. (1989), 22, 601-605], who have advocated the use of a diffractometer-independent definition of the azimuthal angle $\psi$ to specify the diffractiongeometry of a Bragg reflection. It is here proposed that one additional angle $\xi$, which is also based on a diffractometer-independent definition, is needed to encode the direction of linear polarization for those experiments where this quantity is of importance. This definition is then extended to the cases of partially and/or elliptically polarized X-ray beams, and the use of three normalized Stokes parameters, P$_1$, P$_2$ and P$_3$, together with $\xi$, is advocated in order to characterize exhaustively the polarization state of the incident beam. The conventions proposed here present a general, unambiguous and economical means of encoding the information about the diffraction geometry, without the need to record any further information about the instrument, crystal orientation matrix and goniometer angles. Data-processing software using these definitions to analyse polarization-dependent phenomena becomes instrument-independent and completely general. These methods have been implemented in the macromolecular phasing program SHARP for exploiting the polarization anisotropy of anomalous scattering in protein crystals

‘Broken symmetries’ in macromolecular crystallography: phasing from unmerged data

Site-specific radiation damage and anisotropy of anomalous scattering can induce intensity differences in symmetry-related reflections. If the data are kept unmerged, these symmetry-breaking effects can become a source of phase information

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N‐terminal ubiquitin‐like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin‐binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin‐binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin