Project description:A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ? virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.

| S-EPMC4497823 | biostudies-literature

Apoferritin structure at 1.46 angstrom resolution by CryoARM300II equipped with Apollo

Project description: Not available

| EMPIAR-11101 | biostudies-other

2.6A b-Galactosidase Structure Solved Using an Indirect Scintillator-Coupled CMOS Detector at 300 kV

Project description: Not available

| EMPIAR-12033 | biostudies-other

1.8 A resolution structure of β-galactosidase with a 200 kV CRYO ARM electron microscope.

Project description:We report the determination of the structure of Escherichia coli β-galactosidase at a resolution of ∼1.8 Å using data collected on a 200 kV CRYO ARM microscope equipped with a K3 direct electron detector. The data were collected in a single 24 h session by recording images from an array of 7 × 7 holes at each stage position using the automated data collection program SerialEM. In addition to the expected features such as holes in the densities of aromatic residues, the map also shows density bumps corresponding to the locations of hydrogen atoms. The hydrogen densities are useful in assigning absolute orientations for residues such as glutamine or asparagine by removing the uncertainty in the fitting of the amide groups, and are likely to be especially relevant in the context of structure-guided drug design. These findings validate the use of electron microscopes operating at 200 kV for imaging protein complexes at atomic resolution using cryo-EM.

| S-EPMC7340270 | biostudies-literature

Angstrom-resolution fluorescence microscopy.

Project description:Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1-6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7-14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

| S-EPMC10208979 | biostudies-literature

1.8 Å resolution structure of β-galactosidase with a 200 kV CRYO ARM electron microscope

Project description: Not available

| EMPIAR-10446 | biostudies-other

Krios G4 apoferritin test with K3/BioQuantum SerialEM BIS 3x3x1

Project description: Not available

| EMPIAR-10559 | biostudies-other