Atomic-resolution protein structure determination by cryo-EM.

Single-particle electron cryo-microscopy (cryo-EM) is a powerful method for solving the three-dimensional structures of biological macromolecules. The technological development of transmission electron microscopes, detectors and automated procedures in combination with user-friendly image processing software and ever-increasing computational power have made cryo-EM a successful and expanding technology over the past decade1. At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. The direct visualization of atom positions is essential for understanding the mechanisms of protein-catalysed chemical reactions, and for studying how drugs bind to and interfere with the function of proteins2. Here we report a 1.25 Å-resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope that provides, to our knowledge, unprecedented structural detail. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution3). We can visualize individual atoms in a protein, see density for hydrogen atoms and image single-atom chemical modifications. Beyond the nominal improvement in resolution, we also achieve a substantial improvement in the quality of the cryo-EM density map, which is highly relevant for using cryo-EM in structure-based drug design.

Aggregation-induced emissive nanoparticles for fluorescence signaling in a low cost paper-based immunoassay.

Low cost paper based immunoassays are receiving interest due to their fast performance and small amounts of biomolecules needed for developing an immunoassay complex. In this work aggregation-induced emissive (AIE) nanoparticles, obtained from a diastereoisomeric mixture of 1,2-di-(4-hydroxyphenyl)-1,2-diphenylethene (TPEDH) in a one-step top-down method, are characterized through Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Zeta potential. By measuring the Zeta potential before and after labeling the nanoparticles with antibodies we demonstrate that the colloidal system is stable in a wide pH-range. The AIE-active nanoparticles are deposited on chitosan and glutaraldehyde modified paper pads overcoming the common aggregation-caused quenching (ACQ) effect. Analyte concentrations from 1000ng and below are applied in a model immunocomplex using Goat anti-Rabbit IgG and Rabbit IgG. In the range of 7.81ng-250ng, linear trends with a high R(2) are observed, which leads to a strong increase of the blue fluorescence from the TPEDH nanoparticles.