948 research outputs found
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 systÚmes 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 âmultidisciplinary integrationâ). To encourage this multidisciplinary integration, âpreliminary designâ and âdetailed designâ 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 definitionâ 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 âproject-plannedâ method, where project planning is predominant and information is mainly spread out âtop-downâ. 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 expertsâ 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, âthe synergistic combination of mechanical and electrical engineering, computer science, and information technologyâ (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 systĂšmes intĂ©grĂ©s. Ces systĂšmes sont soumis Ă un nombre dâexigences toujours croissant, entraĂźnant des besoins en termes dâintĂ©gration fonctionnelle et spatiale. Ces diffĂ©rents types dâintĂ©gration relative au produit sont Ă©galement la source dâune complexitĂ© organisationnelle, provenant Ă la fois de la multitude dâacteurs rĂ©alisant diffĂ©rentes activitĂ©s dâingĂ©nierie, mais Ă©galement de la diversitĂ© des domaines impliquĂ©s, dĂ©signĂ©e dans ce manuscrit par « intĂ©gration multidisciplinaire ». Pour favoriser cette intĂ©gration multidisciplinaire, les phases de « conception prĂ©liminaire » et de « conception dĂ©taillĂ©e » ont Ă©tĂ© identifiĂ©es comme dĂ©terminantes, notamment car elles se caractĂ©risent par la collaboration de nombreux experts, manipulant un grand nombre de donnĂ©es techniques de dĂ©finition. Les systĂšmes conçus lors de conceptions multidisciplinaires restent faiblement intĂ©grĂ©s. Cela est en partie dĂ» au cloisonnement entre les disciplines et Ă un mode dâorganisation projet basĂ© sur une planification prĂ©dominante, caractĂ©risĂ© notamment par une diffusion de lâinformation principalement descendante (top-down). Afin dâassurer une meilleure collaboration entre ces diffĂ©rentes disciplines, de permettre des prises de dĂ©cision Ă©clairĂ©es par des indicateurs opĂ©rationnels et de pouvoir analyser et mieux comprendre les phĂ©nomĂšnes dâintĂ©gration des expertises, lâintroduction dâune mĂ©thode inspirĂ©e des principes fondateurs des mĂ©thodes agiles est proposĂ©e pour la conception collaborative de systĂšmes intĂ©grĂ©s.La contribution de ces travaux sâappuie sur trois concepts complĂ©mentaires. Le premier, intitulĂ© Collaborative Actions Framework correspond Ă un cadre de collaboration opĂ©rationnelle autour dâactions. Un des objectifs de ce framework est de faciliter la collaboration des acteurs des projets de conception, quelle que soit leur origine disciplinaire, mais Ă©galement dâassurer une traçabilitĂ© entre les prises de dĂ©cision et les corrections/modifications apportĂ©es sur les donnĂ©es techniques. Cette traçabilitĂ© est rendue possible grĂące aux liens existants avec le second concept intitulĂ© Workspace. Apportant un nouvel Ă©clairage sur les possibilitĂ©s offertes par la collaboration autour de ces espaces de collaboration, ce concept offre un certain nombre de possibilitĂ©s,notamment la mise en commun continue des travaux, lâintĂ©gration multidisciplinaire et la validation des modifications. Les Ă©changes de donnĂ©es techniques entre les workspaces, ou le travail simultanĂ© sur les mĂȘmes donnĂ©es techniques, sâappuient quant Ă eux sur la possibilitĂ© de pouvoir gĂ©rer de façon parallĂšle diffĂ©rentes versions dâune mĂȘme donnĂ©e technique. Ces possibilitĂ©s sont proposĂ©es par le troisiĂšme concept, intitulĂ© branch & merge, qui permet Ă©galement Ă diffĂ©rents acteurs de travailler simultanĂ©ment sur les mĂȘmes donnĂ©es. Enfin, ces trois concepts sont ensuite illustrĂ©s par lâintermĂ©diaire dâun dĂ©monstrateur composĂ© dâun scĂ©nario et dâun prototype informatique. Un produit mĂ©catronique, combinaison synergique et systĂ©mique de la mĂ©canique, de l'Ă©lectronique et de l'informatique temps rĂ©el, est utilisĂ© afin dâillustrer les possibilitĂ©s offertes par nos travaux en termes d'intĂ©gration 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 to specify the diffractiongeometry of a Bragg reflection. It is here proposed that one additional angle , 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, P and P, together with , 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
âBroken symmetriesâ 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 Nâterminal ubiquitinâlike (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Ă
crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitinâbinding interface, provide a model for the phosphoubiquitinâparkin 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 ubiquitinâbinding 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
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