Studies in protein crystallography & dynamics, on Membrane Protein Crystallization, the Structure of Thermitase- Eglin and the Apllication of Molecular Dynamics

In dit proefschrift "Onderzoekingen in eiwitkristallografie en dynamica:membraaneiwitten de struktuur van thermitase-egline en de toepassing van moleculaire dynamica"worden de volgende drie onderwerpen besproken. ... Zie: Samenvattin

Cryo-EM structures of peripherin-2 and ROM1 suggest multiple roles in photoreceptor membrane morphogenesis

Mammalian peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) are retina-specific tetraspanins that partake in the constant renewal of stacked membrane discs of photoreceptor cells that enable vision. Here, we present single-particle cryo-electron microscopy structures of solubilized PRPH2-ROM1 heterodimers and higher-order oligomers. High-risk PRPH2 and ROM1 mutations causing blindness map to the protein-dimer interface. Cysteine bridges connect dimers forming positive-curved oligomers, whereas negative-curved oligomers were observed occasionally. Hexamers and octamers exhibit a secondary micelle that envelopes four carboxyl-terminal helices, supporting a potential role in membrane remodeling. Together, the data indicate multiple structures for PRPH2-ROM1 in creating and maintaining compartmentalization of photoreceptor cells

Complement component C3: A structural perspective and potential therapeutic implications

As the most abundant component of the complement system, C3 and its proteolytic derivatives serve essential roles in the function of all three complement pathways. Central to this is a network of protein-protein interactions made possible by the sequential proteolysis and far-reaching structural changes that accompany C3 activation. Beginning with the crystal structures of C3, C3b, and C3c nearly twenty years ago, the physical transformations underlying C3 function that had long been suspected were finally revealed. In the years that followed, a compendium of crystallographic information on C3 derivatives bound to various enzymes, regulators, receptors, and inhibitors generated new levels of insight into the structure and function of the C3 molecule. This Review provides a concise classification, summary, and interpretation of the more than 50 unique crystal structure determinations for human C3. It also highlights other salient features of C3 structure that were made possible through solution-based methods, including Hydrogen/Deuterium Exchange and Small Angle X-ray Scattering. At this pivotal time when the first C3-targeted therapeutics begin to see use in the clinic, some perspectives are also offered on how this continually growing body of structural information might be leveraged for future development of next-generation C3 inhibitors

Improving sampling of crystallographic disorder in ensemble refinement

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallographic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation

Формування політичних інститутів в країнах Центральної Азії в умовах незалежності

Останні досягнення і соціально-економічні успіхи країн Центральної Азії, що отримали незалежність з розпадом СРСР ґрунтуються на багатьох чинниках, серед яких, у першу чергу, проведення ефективних економічних реформ, становлення середнього класу і формування зрілого суспільства

Regulators of complement activity mediate inhibitory mechanisms through a common C3b‐binding mode

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Лексичні дублети у «Російсько-українському словнику» 1924-33 рр.

Lgr5 was originally discovered as a common Wnt target gene in adult intestinal crypts and colon cancer. It was subsequently identified as an exquisite marker of multiple Wnt-driven adult stem cell types. Lgr5 and its homologs, Lgr4 and Lgr6, constitute the receptors for R-spondins, potent Wnt signal enhancers and stem cell growth factors. The Lgr5/R-spondin complex acts by neutralizing Rnf43 and Znrf3, two transmembrane E3 ligases that remove Wnt receptors from the stem cell surface. Rnf43/Znrf3 are themselves encoded by Wnt target genes and constitute a negative Wnt feedback loop. Thus, adult stem cells are controlled by an intricate interplay of potent Wnt agonists, antagonists, and anti-antagonists

Crystal structure of the second extracellular domain of human tetraspanin CD9: twinning and diffuse scattering

Remarkable features are reported in the diffraction pattern produced by a crystal of the second extracellular domain of tetraspanin CD9 (deemed CD9EC2), the structure of which has been described previously [Oosterheert et al. (2020[Oosterheert, W., Xenaki, K. T., Neviani, V., Pos, W., Doulkeridou, S., Manshande, J., Pearce, N. M., Kroon-Batenburg, L. M. J., Lutz, M., van Bergen en Henegouwen, P. M. P. & Gros, P. (2020). Life Sci. Alliance, 3, e202000883.]), Life Sci. Alliance, 3, e202000883]. CD9EC2 crystallized in space group P1 and was twinned. Two types of diffuse streaks are observed. The stronger diffuse streaks are related to the twinning and occur in the direction perpendicular to the twinning interface. It is concluded that the twin domains scatter coherently as both Bragg reflections and diffuse streaks are seen. The weaker streaks along c* are unrelated to the twinning but are caused by intermittent layers of non-crystallographic symmetry related molecules. It is envisaged that the raw diffraction images could be very useful for methods developers trying to remove the diffuse scattering to extract accurate Bragg intensities or using it to model the effect of packing disorder on the molecular structure

Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

Properdin enhances complement-mediated opsonization of targeted cells and particles for immune clearance. Properdin occurs as dimers, trimers and tetramers in human plasma, which recognize C3b-deposited surfaces, promote formation, and prolong the lifetime of C3bBb-enzyme complexes that convert C3 into C3b, thereby enhancing the complement-amplification loop. Here, we report crystal structures of monomerized properdin, which was produced by co-expression of separate N- and C-terminal constructs that yielded monomer-sized properdin complexes that stabilized C3bBb. Consistent with previous low-resolution X-ray and EM data, the crystal structures revealed ring-shaped arrangements that are formed by interactions between thrombospondin type-I repeat (TSR) domains 4 and 6 of one protomer interacting with the N-terminal domain (which adopts a short transforming-growth factor B binding protein-like fold) and domain TSR1 of a second protomer, respectively. Next, a structure of monomerized properdin in complex with the C-terminal domain of C3b showed that properdin-domain TSR5 binds along the C-terminal a-helix of C3b, while two loops, one from domain TSR5 and one from TSR6, extend and fold around the C3b C-terminus like stirrups. This suggests a mechanistic model in which these TSR5 and TSR6 "stirrups" bridge interactions between C3b and factor B or its fragment Bb, and thereby enhance formation of C3bB pro-convertases and stabilize C3bBb convertases. In addition, properdin TSR6 would sterically block binding of the protease factor I to C3b, thus limiting C3b proteolytic degradation. The presence of a valine instead of a third tryptophan in the canonical Trp-ladder of TSR domains in TSR4 allows a remarkable ca. 60 degrees-domain bending motion of TSR4. Together with variable positioning of TSR2 and, putatively, TSR3, this explains the conformational flexibility required for properdin to form dimers, trimers, and tetramers. In conclusion, the results indicate that binding avidity of oligomeric properdin is needed to distinguish surface-deposited C3b molecules from soluble C3b or C3 and suggest that properdin-mediated interactions bridging C3b-B and C3b-Bb enhance affinity, thus promoting convertase formation and stabilization. These mechanisms explain the enhancement of complement-mediated opsonization of targeted cells and particle for immune clearance