Fully automated, sequential tilt-series acquisition with Leginon

Electron tomography has become a uniquely powerful tool for investigating the structures of individual cells, viruses, and macromolecules. Data collection is, however, time consuming and requires expensive instruments. To optimize productivity, we have incorporated one of the existing tilt-series acquisition programs, UCSF Tomo, into the well-developed automatic electron microscopy data collection package Leginon to enable fully automatic, sequential tilt-series acquisition. Here we describe how UCSF Tomo was integrated into Leginon, what users must do to set up a data collection session, how the automatic collection proceeds, how archived data about the process can be accessed and used, and how the software has been tested

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography

Open Access funded by Medical Research CouncilThe MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages. Following extensive discussions, the authors, who represent leading software packages in the field, propose a set of extensions to the MRC format standard designed to accommodate these variants, while restoring interoperability. The MRC format is equivalent to the map format used in the CCP4 suite for macromolecular crystallography, and the proposal also maintains interoperability with crystallography software. This Technical Note describes the proposed extensions, and serves as a reference for the standard.We thank Chris Booth and Steffen Meyer from Gatan Inc. for clarifying the format definition used by Digital Micrograph. Acknowledgement for support from National Institute of Health, USA includes: NIGMS grant P41GM103310 (AC and SD), NIBIB grant 5R01-EB005027 (DM), and R01GM080139 (SJL). RH and MW would like to thank the UK Medical Research Council for the award of Partnership Grant MR/J000825/1 to support the establishment of CCP-EM. RH and JS are also supported by MRC grant U105184322

Structure-based vaccine design by electron microscopy

Modern vaccine design relies on multiscale, interdisciplinary efforts that take advantage of innovative technologies such as in silico identification of antigens, high throughput screening of antigen immunogenicity, and gene expression profiling to predict host immune responses. In recent years, structural analysis has played an increasingly important role in vaccine development as a means to improve antigen stability, immunogenicity and large scale production. Transmission electron microscopy (TEM), and in particular cryo-TEM, is an established and powerful imaging technique applicable to many specimens, including the three-dimensional (3D) reconstruction of macromolecules and their associated complexes to high resolution. The technique is parsimonious in its material requirements and captures the specimens in their fully hydrated state, close to their native environment. The resolution of cryo-TEM reconstructions was limited to the subnanometer range until the recent development of direct electron detectors and improvements in image processing software, which has led to a so-called “resolution revolution” in the cryo-TEM field. Several protein structures have now been solved at near atomic resolution, establishing the technique as a viable alternative to X-ray analysis for high resolution structure determination. We have determined several structures with and without bound compounds at 2.9 Å – 3.6 Å resolution, which are being integrated into drug discovery and development workflows by our clients. Here we present the 2.4Å resolution structure of apoferritin determined with our Titan Krios electron microscope as an example of the cryo-TEM services available at NIS. These services are significantly enhanced with unique access by NIS to a new instrument, Spotiton, a robotic device that dispenses picoliter-volumes of sample onto a self-blotting nanowire grid as it flies past en route to vitrification. This provides several advantages over standard vitrification methods, including more automated and reproducible preparation of specimens and reducing the deleterious effects of particles interacting with the air-water interface. While high resolution 3D structure determination by cryo-TEM is at the forefront of structural biology, averages of 2D projection images at moderate resolution in negative stain or vitreous ice can also provide a wealth of information that may be difficult to obtain using other methods. This is illustrated in a number of case studies, including (1) mapping of neutralizing epitopes on the CMV pentameric glycoprotein complex; (2) mapping of neutralizing epitopes on the HIV-1 envelope glycoprotein trimer; (3) assessment of structure and conformational stability of pre- and post-fusion RSV-F protein; (4) characterization of novel adjuvants and protein delivery systems. In summary, both the moderate resolution TEM and high resolution cryo-TEM methods are well suited to extensively characterize antigen structure-function relationships, some of which may be refractory to other experimental methods

Routine single particle CryoEM sample and grid characterization by tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment

An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy

A new software package allows for dense electron microscopy reconstructions of neuronal networks in the fruit fly brain, and reveals specific differences in microcircuits between insects and vertebrates

Automated data collection in single particle electron microscopy

10.1093/jmicro/dfv369MICROSCOPY65143-5