Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene.

Cryogenic electron microscopy can provide high-resolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built de novo. However, the interaction between the ionizing electron beam and the sample results in beam-induced motion and image distortion, which limit the attainable resolutions. Sample charging is one contributing factor of beam-induced motions and image distortions, which is normally alleviated by including part of the supporting conducting film within the beam-exposed region. However, routine data collection schemes avoid strategies whereby the beam is not in contact with the supporting film, whose rationale is not fully understood. Here we characterize electrostatic charging of vitreous samples, both in imaging and in diffraction mode. We mitigate sample charging by depositing a single layer of conductive graphene on top of regular EM grids. We obtained high-resolution single-particle analysis (SPA) reconstructions at 2 Å when the electron beam only irradiates the middle of the hole on graphene-coated grids, using data collection schemes that previously failed to produce sub 3 Å reconstructions without the graphene layer. We also observe that the SPA data obtained with the graphene-coated grids exhibit a higher b factor and reduced particle movement compared to data obtained without the graphene layer. This mitigation of charging could have broad implications for various EM techniques, including SPA and cryotomography, and for the study of radiation damage and the development of future sample carriers. Furthermore, it may facilitate the exploration of more dose-efficient, scanning transmission EM based SPA techniques.

Single-particle cryo-EM: alternative schemes to improve dose efficiency.

Imaging of biomolecules by ionizing radiation, such as electrons, causes radiation damage which introduces structural and compositional changes of the specimen. The total number of high-energy electrons per surface area that can be used for imaging in cryogenic electron microscopy (cryo-EM) is severely restricted due to radiation damage, resulting in low signal-to-noise ratios (SNR). High resolution details are dampened by the transfer function of the microscope and detector, and are the first to be lost as radiation damage alters the individual molecules which are presumed to be identical during averaging. As a consequence, radiation damage puts a limit on the particle size and sample heterogeneity with which electron microscopy (EM) can deal. Since a transmission EM (TEM) image is formed from the scattering process of the electron by the specimen interaction potential, radiation damage is inevitable. However, we can aim to maximize the information transfer for a given dose and increase the SNR by finding alternatives to the conventional phase-contrast cryo-EM techniques. Here some alternative transmission electron microscopy techniques are reviewed, including phase plate, multi-pass transmission electron microscopy, off-axis holography, ptychography and a quantum sorter. Their prospects for providing more or complementary structural information within the limited lifetime of the sample are discussed.

Sub-pixel electron detection using a convolutional neural network.

Modern direct electron detectors (DEDs) provided a giant leap in the use of cryogenic electron microscopy (cryo-EM) to study the structures of macromolecules and complexes thereof. However, the currently available commercial DEDs, all based on the monolithic active pixel sensor, still require relative long exposure times and their best results have only been obtained at 300 keV. There is a need for pixelated electron counting detectors that can be operated at a broader range of energies, at higher throughput and higher dynamic range. Hybrid Pixel Detectors (HPDs) of the Medipix family were reported to be unsuitable for cryo-EM at energies above 80 keV as those electrons would affect too many pixels. Here we show that the Timepix3, part of the Medipix family, can be used for cryo-EM applications at higher energies. We tested Timepix3 detectors on a 200 keV FEI Tecnai Arctica microscope and a 300 keV FEI Tecnai G2 Polara microscope. A correction method was developed to correct for per-pixel differences in output. Timepix3 data were simulated for individual electron events using the package Geant4Medipix. Global statistical characteristics of the simulated detector response were in good agreement with experimental results. A convolutional neural network (CNN) was trained using the simulated data to predict the incident position of the electron within a pixel cluster. After training, the CNN predicted, on average, 0.50 pixel and 0.68 pixel from the incident electron position for 200 keV and 300 keV electrons respectively. The CNN improved the MTF of experimental data at half Nyquist from 0.39 to 0.70 at 200 keV, and from 0.06 to 0.65 at 300 keV respectively. We illustrate that the useful dose-lifetime of a protein can be measured within a 1 second exposure using Timepix3.

Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo.

During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.