Direct electron detection camera technology for transmission electron microscopy has provided a dramatic improvement in the quality of images that can be acquired in cryo-electron microscopy (cryo-EM). Improvement in image quality is one of the key factors that has led to the resolution revolution 1 so that reconstructions of biological macromolecules are now commonly resolved to a resolution better than 3.5 Angstroms. Although this resolution revolution opens new opportunities for cryo-EM to solve key problems in molecular biology 2 , it remains a challenge to increase imaging throughput while maintaining the highest image quality. This is primarily due to the numerous inter-related microscope and camera parameters that need to be carefully optimized to balance between imaging efficiency and image quality. Optimization of these parameters is of critical importance for biopharmaceutical research since both image quality and throughput must be maximized 3 .

The adenosine A1 receptor (A1R) is a key mediator of the neuroprotective effect by endogenous adenosine. Yet targeting this receptor for neuroprotection is challenging due to its broad expression throughout the body. A mechanistic understanding of the regulation of A1R signaling is necessary for the future design of therapeutic agents that can selectively enhance A1R-mediated responses in the nervous system. In this study, we demonstrate that A1R activation leads to a sustained localization of regulator of G protein signaling 4 (RGS4) at the plasmamembrane, a process that requires neurabin (a neural tissue-specific protein). A1R and RGS4 interact with the overlapping regions of neurabin. In addition, neurabin domains required for oligomerization are essential for formation of the A1R/neurabin/RGS4 ternary complex, as well as for stable localization of RGS4 at the plasma membrane and attenuation of A1R signaling. Thus, A1R and RGS4 each likely interact with one neurabin molecule in a neurabin homo-oligomer to form a ternary complex, representing a novel mode of regulation of G protein-coupled receptor signaling by scaffolding proteins. Our mechanistic analysis of neurabin-mediated regulation of A1R signaling in this study will be valuable for the future design of therapeutic agents that can selectively enhance A1R-mediated responses in the nervous system.

Journal Article Plasma Cleaning Improves the Image Quality of Serial Block-face Scanning Electron Microscopy (SBFSEM) Volumetric Data Sets Get access Barbara Armbruster, Barbara Armbruster XEI Scientific, Inc., Redwood City, CA, USA Search for other works by this author on: Oxford Academic Google Scholar Christopher Booth, Christopher Booth Gatan, Inc., Pleasanton, CA, USA Search for other works by this author on: Oxford Academic Google Scholar Stuart Searle, Stuart Searle Gatan UK, Abingdon, Oxon, United Kingdom Search for other works by this author on: Oxford Academic Google Scholar Michael Cable, Michael Cable XEI Scientific, Inc., Redwood City, CA, USA Search for other works by this author on: Oxford Academic Google Scholar Ronald Vane Ronald Vane XEI Scientific, Inc., Redwood City, CA, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 23, Issue S1, 1 July 2017, Pages 1266–1267, https://doi.org/10.1017/S1431927617006997 Published: 04 August 2017

Cryo-electron microscopy has seen incredible progress recently. From the first reconstruction of frozen hydrated samples to recent atomicresolution structures, structural biology is undergoing a revolution. However, until recently, the resolution of cryo-EM structures was often significantly lower than that of X-ray crystallography. We have reached a point where, for an increasing number of samples, the resolution achieved using the two techniques are comparable. Atomic scale resolution in cryo-TEM is now possible with the introduction of electron-counting direct detection cameras. We review the progress in the technology of TEM cameras and describe an example of how cutting edge electron counting cameras are being applied in high-resolution cryo-

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., section loss and image distortions. New methods, such as serial block-face scanning electron microscopy (SBFSEM) overcome these limitations and promise a high lateral resolution. However, little is known about the usability of SBFSEM in diminutive, but highly complex cellular systems. We used spider sperm (~3 µm in diameter), which fulfills these conditions, to analyze the potential of SBFSEM compared with ssTEM. Our data suggest that the resolution obtained by SBFSEM allows depicting structures on a cellular level and is sufficient to discriminate subcellular components, but is highly dependent on previous staining procedures and electron density of the target structures.

The combination of a direct electron-detection camera that can count individual electrons and an algorithm for correcting for beam-induced motion in cryo-EM will facilitate determination of three-dimensional structures of smaller, lower-symmetry macromolecular complexes to higher resolution than previously possible. 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). Using this approach, we determined a 3.3-Å-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency—key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

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. In this paper, we describe how JADAS is used to accurately locate and image suitable specimen areas on a grid of ice-embedded particles. We also demonstrate the utility of JADAS by imaging the epsilon 15 bacteriophage with the JEM3200FSC electron cryo-microscope, showing that sufficient images can be collected in a single 8h session to yield a subnanometer resolution structure which agrees with the previously determined structure.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

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° rotation to minimize artifacts. The end result is a four-dimensional data set, containing two spatial dimensions, rotation angle and energy-loss information I(x, y, θ, ΔE), which can then be processed to extract any EELS signal as a rotation or “tilt-series” map. If the extracted properties satisfy the linear projection criteria, these maps can then be used for tomographic reconstruction to yield volumetric maps of the corresponding properties. Hence by combining STEM HAADF and energy-loss information from such a series of spectrum images, it is possible to map not only the microstructure, but also the elemental, physical and chemical state information of a material in three dimensions. Two examples are reported here to demonstrate the potential of this technique. To illustrate chemical tomography, 4D STEM-EELS was used to directly probe the 3D electronic structure of a W-to-Si contact from a semiconductor device. Core-loss data were used to reconstruct and render the composition of the W-to-Si contact in three dimensions. The fine structure of the 99eV Si edge was analyzed with MLLS fitting to map the variations in Si bonding in 3D. To illustrate the direct probing of intrinsic material anisotropy, 4D STEM-EELS was used to probe a ZnO thin film. Subtle but systematic changes in low-loss structure were observed as a function of electron-beam orientation with respect to the ZnO crystallographic axes. Together these examples illustrate how the 4D STEM-EELS technique reported here can be used to probe the elemental, physical and chemical state information of a material in three dimensions and extend our knowledge of nano-scale structures.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

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. Comparison of TRiC in its open, nucleotide-free, and closed, nucleotide-induced states reveals that the interdomain motions leading to lid closure in TRiC are radically different from those of prokaryotic chaperonins, despite their overall structural similarity. We propose that domain movements in TRiC are coordinated through unique interdomain contacts within each subunit and, further, these contacts are absent in prokaryotic chaperonins. Our findings show how different mechanical switches can evolve from a common structural framework through modification of allosteric networks.

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. This approach depends neither on a preexisting template, nor an exact knowledge of the geometry, orientation, or amount of the missing data. This paper introduces a procedure where the missing data might be gradually "filled in" by consecutively aligning and averaging volumes with different orientations of their missing data. We have validated these techniques by a set of simulated data with various symmetries and extent of missing data. We have also successfully applied these procedures to experimental cryo-electron tomographic data [Chang, J.T., Schmid, M.F., Rixon, F.J., and Chiu, W., 2007. Electron cryotomography reveals the portal in the herpesvirus capsid. J. Virol. 81, 2065–2068; Schmid, M.F., Paredes, A.M., Khant, H.A., Soyer, F., Aldrich, H.C., Chiu, W., and Shively, J.M., 2006. Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography. J. Mol. Biol. 364, 526–535].

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. We have used the JAMES API INTEGRATOR to develop two applications for low-dose digital imaging: JAMES imaging application and the mr T tomographic imaging application. Both applications have been widely used within our NCRR-supported Center for routine data collection and are now made available for public download.

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. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.

Journal Article Moving Beyond Bright Field Transmission Electron Microscopy Get access CR Booth, CR Booth Gatan Inc Search for other works by this author on: Oxford Academic Google Scholar RT Harmon, RT Harmon Gatan Inc Search for other works by this author on: Oxford Academic Google Scholar S Meyer, S Meyer Gatan Inc Search for other works by this author on: Oxford Academic Google Scholar RD Twesten, RD Twesten Gatan Inc Search for other works by this author on: Oxford Academic Google Scholar I Arslan, I Arslan Sandia National Laboratories Search for other works by this author on: Oxford Academic Google Scholar JP Bradley, JP Bradley Lawrence Livermore National Laboratory Search for other works by this author on: Oxford Academic Google Scholar MA Aronova, MA Aronova National Institutes of Health Search for other works by this author on: Oxford Academic Google Scholar RD Leapman, RD Leapman National Institutes of Health Search for other works by this author on: Oxford Academic Google Scholar JA Hunt JA Hunt Gatan Inc Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 13, Issue S02, 1 August 2007, Pages 1320–1321, https://doi.org/10.1017/S1431927607076957 Published: 05 August 2007

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. Using this parameter, we demonstrate that the SNR of images of either amorphous carbon film or ice embedded virus particles collected on a new commercially available 4kx4k CCD camera are slightly better than photographic film at low spatial frequency (<1/5 Nyquist frequency), and as good as photographic film out to half of the Nyquist frequency. In addition it is slightly easier to visualize ice embedded particles on this CCD camera than on photographic film. Based on this analysis it is realistic to collect images containing subnanometer resolution data (6–9Å) using this CCD camera at an effective magnification of ∼112000× on a 300kV electron microscope.