Serial block-face imaging and its potential for reconstructing diminutive cell systems: a case study from arthropods.

Until recently, three-dimensional reconstruction on an ultrastructural level was only possible using serial section transmission electron microscopy (ssTEM). However, ssTEM is highly challenging and prone to artifacts as, e.g.

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM.

In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 Å).

Four-dimensional STEM-EELS: enabling nano-scale chemical tomography.

Advances in electron-based instrumentation have enabled the acquisition of multidimensional data sets for exploring the unique structure-property relationship of nanomaterials. In this manuscript, we report a technique for directly probing and analyzing the three-dimensional (3D) electronic structure of a material at the nano-scale. This technique, referred to here as 4D STEM-EELS, utilizes a rotation holder and pillar-shaped samples to allow STEM mode high-angle annular dark-field (HAADF) and EELS spectrum images to be recorded over a complete 180 degrees rotation to minimize artifacts.

JADAS: a customizable automated data acquisition system and its application to ice-embedded single particles.

The JEOL Automated Data Acquisition System (JADAS) is a software system built for the latest generation of the JEOL Transmission Electron Microscopes. It is designed to partially or fully automate image acquisition for ice-embedded single particles under low dose conditions. Its built-in flexibility permits users to customize the order of various imaging operations.

Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT.

All chaperonins mediate ATP-dependent polypeptide folding by confining substrates within a central chamber. Intriguingly, the eukaryotic chaperonin TRiC (also called CCT) uses a built-in lid to close the chamber, whereas prokaryotic chaperonins use a detachable lid. Here we determine the mechanism of lid closure in TRiC using single-particle cryo-EM and comparative protein modeling.

Methods for aligning and for averaging 3D volumes with missing data.

The visibility and resolution of a tomographic reconstruction containing multiple copies of discrete particles can be enhanced by averaging subtomograms after they are corrected aligned. However, the "missing wedge" in electron tomography can easily lead to erroneous alignment. We have explored a Fourier space cross-correlation method with a proper weighting scheme to align and average different sets of volumetric data, each of which has different missing data due to the limited specimen tilts.

Modular software platform for low-dose electron microscopy and tomography.

Transmission electron microscopy imaging protocols required by structural scientists vary widely and can be laborious without tailor-made applications. We present here the jeol automated microscopy expert system (james) api integrator, a programming library for computer control of transmission electron microscopy operations and equipment. james has been implemented on JEOL microscopes with Gatan CCDs but is designed to be modular so it can be adapted to run on different microscopes and detectors.

Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins.

Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged.

Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300kV.

CCD cameras have numerous advantages over photographic film for detecting electrons; however the point spread function of these cameras has not been sufficient for single particle data collection to subnanometer resolution with 300kV microscopes. We have adopted spectral signal to noise ratio (SNR) as a parameter for assessing detector quality for single particle imaging. The robustness of this parameter is confirmed under a variety of experimental conditions.

Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase composed of approximately 13 distinct subunits required for progression through meiosis, mitosis, and the G1 phase of the cell cycle. Despite its central role in these processes, information concerning its composition and structure is limited. Here, we determined the structure of yeast APC/C by cryo-electron microscopy (cryo-EM).

A structure of the human apoptosome at 12.8 A resolution provides insights into this cell death platform.

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods.

A 9 angstroms single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope.

Sub-nanometer resolution structure determination is becoming a common practice in electron cryomicroscopy of macromolecular assemblies. The data for these studies have until now been collected on photographic film. Using cytoplasmic polyhedrosis virus (CPV), a previously determined structure, as a test specimen, we show the feasibility of obtaining a 9 angstroms structure from images acquired from a 4 k x 4 k Gatan CCD on a 200 kV electron cryomicroscope.