Maryam Khoshouei

Volta potential phase plate for in-focus phase contrast transmission electron microscopy

Significance Biological electron cryomicroscopy is limited by the radiation sensitivity of the samples and the consequent need to minimize exposure to the beam. This, in turn, results in low-contrast images with a poor signal-to-noise ratio. The current practice to improve phase contrast by defocusing results in contrast transfer functions necessitating image restoration to provide interpretable data. Phase plates enable in-focus phase contrast, but the existing ones, including the thin film Zernike-type phase plate, suffer from severe limitations, such as a short usable life span, fringing artifacts, and problems in using them in automated data acquisition procedures. The Volta phase plate presented here solves those problems and has the potential to become a practical solution for in-focus phase contrast in transmission electron microscopy. We describe a phase plate for transmission electron microscopy taking advantage of a hitherto-unknown phenomenon, namely a beam-induced Volta potential on the surface of a continuous thin film. The Volta potential is negative, indicating that it is not caused by beam-induced electrostatic charging. The film must be heated to ∼200 °C to prevent contamination and enable the Volta potential effect. The phase shift is created “on the fly” by the central diffraction beam eliminating the need for precise phase plate alignment. Images acquired with the Volta phase plate (VPP) show higher contrast and unlike Zernike phase plate images no fringing artifacts. Following installation into the microscope, the VPP has an initial settling time of about a week after which the phase shift behavior becomes stable. The VPP has a long service life and has been used for more than 6 mo without noticeable degradation in performance. The mechanism underlying the VPP is the same as the one responsible for the degradation over time of the performance of thin-film Zernike phase plates, but in the VPP it is used in a constructive way. The exact physics and/or chemistry behind the process causing the Volta potential are not fully understood, but experimental evidence suggests that radiation-induced surface modification combined with a chemical equilibrium between the surface and residual gases in the vacuum play an important role.

Phase-plate cryo-EM structure of a class B GPCR–G-protein complex

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsβγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with the intrinsically low contrast and beam-induced motion of radiation-sensitive organic materials embedded in amorphous ice, and hence they have enabled the structure determination of many macromolecular assemblies to atomic or near-atomic resolution. Nevertheless, there are still limitations and one of them is the size of the target structure. Here, we report the use of a Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 Å. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.

Volta phase plate cryo-EM of the small protein complex Prx3

Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors. However, sub-nanometre resolution cryo-EM structures are rare compared with crystal structure depositions, particularly for relatively small particles (<400 kDa). Here we demonstrate the benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure determination of 257 kDa human peroxiredoxin-3 dodecamers at 4.4 Å resolution. The Volta phase plate improves the applicability of cryo-EM for small molecules and accelerates structure determination.

Revisiting the Structure of Hemoglobin and Myoglobin with Cryo-Electron Microscopy

Sixty years ago, the first protein structure of myoglobin was determined by John Kendrew and his colleagues; hemoglobin followed shortly thereafter. For quite some time, it seemed that only X-ray crystallography would be capable of determining the structure of proteins to high resolution. In recent years, cryo-electron microscopy has emerged as a viable alternative and indeed in many cases the preferred approach. It is capable of studying proteins that span a size range from several megadaltons to proteins as small as myoglobin and hemoglobin.

Isolation and Characterization of Metallosphaera Turreted Icosahedral Virus, a Founding Member of a New Family of Archaeal Viruses

ABSTRACT Our understanding of archaeal virus diversity and structure is just beginning to emerge. Here we describe a new archaeal virus, tentatively named Metallosphaera turreted icosahedral virus (MTIV), that was isolated from an acidic hot spring in Yellowstone National Park, USA. Two strains of the virus were identified and were found to replicate in an archaeal host species closely related to Metallosphaera yellowstonensis. Each strain encodes a 9.8- to 9.9-kb linear double-stranded DNA (dsDNA) genome with large inverted terminal repeats. Each genome encodes 21 open reading frames (ORFs). The ORFs display high homology between the strains, but they are quite distinct from other known viral genes. The 70-nm-diameter virion is built on a T=28 icosahedral lattice. Both single particle cryo-electron microscopy and cryotomography reconstructions reveal an unusual structure that has 42 turret-like projections: 12 pentameric turrets positioned on the icosahedral 5-fold axes and 30 turrets with apparent hexameric symmetry positioned on the icosahedral 2-fold axes. Both the virion structural properties and the genome content support MTIV as the founding member of a new family of archaeal viruses. IMPORTANCE Many archaeal viruses are quite different from viruses infecting bacteria and eukaryotes. Initial characterization of MTIV reveals a virus distinct from other known bacterial, eukaryotic, and archaeal viruses; this finding suggests that viruses infecting Archaea are still an understudied group. As the first known virus infecting a Metallosphaera sp., MTIV provides a new system for exploring archaeal virology by examining host-virus interactions and the unique features of MTIV structure-function relationships. These studies will likely expand our understanding of virus ecology and evolution.

Phase Contrast Single Particle Analysis at Atomic Resolutions

Single particle analysis and X-ray crystallography are two standard approaches to answer many biological questions. However, X-ray crystallography has its own limitations due to non-sufficient crystal size or non-diffracted crystals. In this case single particle analysis become a strong complementary tool to deliver a wealth of structural information. In this approach the samples are highly purified in solution and plunge frozen in liquid ethane or ethane propane mixture which is cooled down by liquid nitrogen. Moreover, the samples embedded in a thin layer of vitreous ice leads to poor contrast due to low signal-to-noise ratio. There are two ways of generating phase contrast from the weak phase objects in their native and frozen hydrated states: one way is using the spherical aberration of the microscope lenses and intended defocus of the objective lens and the other way is using a phase plate. By using the phase plates, the low frequency information is well and continuously transferred to high frequency information, which is the strength of the phase contrast method compared to defocused based cryo-electron microscopy.

Towards High Resolution in Cryo-Electron Tomography Subtomogram Analysis

Cryo-electron tomography allows three-dimensional imaging of macromolecular complexes in a closeto-native state at molecular resolution [1, 2]. Due to specimen damage imposed by the imaging electrons, the signal-to-noise-ratio in cryo-electron tomography data is typically very low. This low signal-to-noise-ratio restricts interpretability of raw cryo-electron tomograms, but can ultimately be overcome by averaging many subtomograms depicting the same macromolecular complex [2-4]. Here, we introduce several approaches that have been useful to deal with the low signal-to-noise ratio and to overcome other resolution limiting factors in subtomogram analysis that mostly originate from tilting of the specimen during data acquisition.

CryoEM structure determination of small protein complexes with the Volta phase plate

Phase contrast microscopy has been used in light microscopy for imaging unstained biological material for more than 80 years, opens up new possibilities in structural biology by cryo-electron microscopy (cryoEM). Images of unstained and frozen-hydrated biological material produce very little contrast, since such samples interact weakly with high-energy electrons. The only way to produce contrast in cryo-EM (until recently) was to intentionally defocus the image at the expense of altering high-resolution structural information. Despite the various efforts made in the development of EM phase plates, the technique was not readily applicable for cryo-EM, mainly due to the very short ‘life time’ of a phase plate and its complicated use.

GPCR activation: an intertwined history of crystallography and EM

G-protein coupled receptors (GPCRs) remain the most successful class of drug targets. However, only a small subset of human GPCR structures have been solved to date. Of those, GPCRs in complex with the effector G-protein are particularly recalcitrant to crystallisation. Single particle EM played a key role in determining the crystal structure of a ligand-receptor-G-protein ternary complex [1,2]. Advances in phase plate cryo-EM have shown that high-resolution structure determination of small protein complexes is facile by single particle analysis [3]. Here, we present a staged development process for structure determination of intricate membrane protein complexes, highlighting complementary strengths of crystallography and cryo-EM.