STRUCTURE DETERMINATION OF HETEROGENEOUS BIOLOGICAL SPECIMENS IN CROWDED ENVIRONMENTS

The central dogma of molecular biology describes a strictly linear flow of genetic information stored in DNA transferred through RNA and translated into protein products. In the “post-genomic era” however, it is evident that abundant information flows from protein to protein and even protein back to DNA. The field of Structural Biology seeks to understand how the spatial and temporal organization of that information is stored and transmitted via the three-dimensional structure and dynamics of biological macromolecules. X-ray crystallography, nuclear magnetic resonance, and single particle cryo-electron microscopy (cryo-EM) are the primary techniques available to the structural biologist to deduce structure and dynamics at or near atomic resolutions. These tools are generally limited to the study of stable molecules that can be purified biochemically. Other approaches, like super-resolution light microscopy and cryo-electron tomography (cryo-ET), are amenable to the study of more labile macromolecular complexes or those found in situ; however, they are limited to resolutions of tens of nanometers. Improving the resolving capability of cryo-ET with sub-tomogram averaging to routinely reach beyond 10 Å is the primary goal of this work. My unique contribution to the field of structural biology is a suite of software tools called emClarity (enhanced macromolecular classification and alignment for high-resolution in situ tomography) which allows scientists with minimal computational background to probe the structural states of conformationally variable molecules present in complex and crowded environments

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand

National synchrotron light source. Activity report, October 1, 1995--September 30, 1996

The hard work done by the synchrotron radiation community, in collaboration with all those using large-scale central facilities during 1995, paid off in FY 1996 through the DOE`s Presidential Scientific Facilities Initiative. In comparison with the other DOE synchrotron radiation facilities, the National Synchrotron Light Source benefited least in operating budgets because it was unable to increase running time beyond 100%-nevertheless, the number of station hours was maintained. The major thrust at Brookhaven came from a 15% increase in budget which allowed the recruitment of seven staff in the beamlines support group and permitted a step increment in the funding of the extremely long list of upgrades; both to the sources and to the beamlines. During the December 1995 shutdown, the VUV Ring quadrant around U10-U12 was totally reconstructed. New front ends, enabling apertures up to 90 mrad on U10 and U12, were installed. During the year new PRTs were in formation for the infrared beamlines, encouraged by the investment the lab was able to commit from the initiative funds and by awards from the Scientific Facilities Initiative. A new PRT, specifically for small and wide angle x-ray scattering from polymers, will start work on X27C in FY 1997 and existing PRTs on X26C and X9B working on macromolecular crystallography will be joined by new members. Plans to replace aging radio frequency cavities by an improved design, originally a painfully slow six or eight year project, were brought forward so that the first pair of cavities (half of the project for the X-Ray Ring) will now be installed in FY 1997. Current upgrades to 350 mA initially and to 438 mA later in the X-Ray Ring were set aside due to lack of funds for the necessary thermally robust beryllium windows. The Scientific Facilities Initiative allowed purchase of all 34 windows in FY 1996 so that the power upgrade will be achieved in FY 1997

[¹⁸F]fluorination of biorelevant arylboronic acid pinacol ester scaffolds synthesized by convergence techniques

Aim: The development of small molecules through convergent multicomponent reactions (MCR) has been boosted during the last decade due to the ability to synthesize, virtually without any side-products, numerous small drug-like molecules with several degrees of structural diversity.(1) The association of positron emission tomography (PET) labeling techniques in line with the “one-pot” development of biologically active compounds has the potential to become relevant not only for the evaluation and characterization of those MCR products through molecular imaging, but also to increase the library of radiotracers available. Therefore, since the [18F]fluorination of arylboronic acid pinacol ester derivatives tolerates electron-poor and electro-rich arenes and various functional groups,(2) the main goal of this research work was to achieve the 18F-radiolabeling of several different molecules synthesized through MCR. Materials and Methods: [18F]Fluorination of boronic acid pinacol esters was first extensively optimized using a benzaldehyde derivative in relation to the ideal amount of Cu(II) catalyst and precursor to be used, as well as the reaction solvent. Radiochemical conversion (RCC) yields were assessed by TLC-SG. The optimized radiolabeling conditions were subsequently applied to several structurally different MCR scaffolds comprising biologically relevant pharmacophores (e.g. β-lactam, morpholine, tetrazole, oxazole) that were synthesized to specifically contain a boronic acid pinacol ester group. Results: Radiolabeling with fluorine-18 was achieved with volumes (800 μl) and activities (≤ 2 GBq) compatible with most radiochemistry techniques and modules. In summary, an increase in the quantities of precursor or Cu(II) catalyst lead to higher conversion yields. An optimal amount of precursor (0.06 mmol) and Cu(OTf)2(py)4 (0.04 mmol) was defined for further reactions, with DMA being a preferential solvent over DMF. RCC yields from 15% to 76%, depending on the scaffold, were reproducibly achieved. Interestingly, it was noticed that the structure of the scaffolds, beyond the arylboronic acid, exerts some influence in the final RCC, with electron-withdrawing groups in the para position apparently enhancing the radiolabeling yield. Conclusion: The developed method with high RCC and reproducibility has the potential to be applied in line with MCR and also has a possibility to be incorporated in a later stage of this convergent “one-pot” synthesis strategy. Further studies are currently ongoing to apply this radiolabeling concept to fluorine-containing approved drugs whose boronic acid pinacol ester precursors can be synthesized through MCR (e.g. atorvastatin)