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4 Publications

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    Grigorieff Lab
    12/01/13 | Quantitative characterization of electron detectors for transmission electron microscopy.
    Ruskin RS, Yu Z, Grigorieff N
    Journal of Structural Biology. 2013 Dec;184(3):385-93. doi: 10.1016/j.jsb.2013.10.016

    A new generation of direct electron detectors for transmission electron microscopy (TEM) promises significant improvement over previous detectors in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE). However, the performance of these new detectors needs to be carefully monitored in order to optimize imaging conditions and check for degradation over time. We have developed an easy-to-use software tool, FindDQE, to measure MTF and DQE of electron detectors using images of a microscope’s built-in beam stop. Using this software, we have determined the DQE curves of four direct electron detectors currently available: the Gatan K2 Summit, the FEI Falcon I and II, and the Direct Electron DE-12, under a variety of total dose and dose rate conditions. We have additionally measured the curves for the Gatan US4000 and TVIPS TemCam-F416 scintillator-based cameras. We compare the results from our new method with published curves.

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    Grigorieff Lab
    09/01/13 | Likelihood-based classification of cryo-EM images using FREALIGN.
    Lyumkis D, Brilot AF, Theobald DL, Grigorieff N
    Journal of Structural Biology. 2013 Sep;183(3):377-88. doi: 10.1016/j.jsb.2013.07.005

    We describe an implementation of maximum likelihood classification for single particle electron cryo-microscopy that is based on the FREALIGN software. Particle alignment parameters are determined by maximizing a joint likelihood that can include hierarchical priors, while classification is performed by expectation maximization of a marginal likelihood. We test the FREALIGN implementation using a simulated dataset containing computer-generated projection images of three different 70S ribosome structures, as well as a publicly available dataset of 70S ribosomes. The results show that the mixed strategy of the new FREALIGN algorithm yields performance on par with other maximum likelihood implementations, while remaining computationally efficient.

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    Grigorieff Lab
    02/19/13 | Direct detection pays off for electron cryo-microscopy.
    Grigorieff N
    eLife. 2013 Feb 19;2:e00573. doi: 10.7554/eLife.00573

    Improved electron detectors and image-processing techniques will allow the structures of macromolecules to be determined from tens of thousands of single-particle cryo-EM images, rather than the hundreds of thousands needed previously.

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    Grigorieff Lab
    01/09/13 | Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles.
    Estrozi LF, Settembre EC, Goret G, McClain B, Zhang X, Chen JZ, Grigorieff N, Harrison SC
    Journal of Molecular Biology. 2013 Jan 9;425(1):124-32. doi: 10.1016/j.jmb.2012.10.011

    Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes. Analysis of the X-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the X-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the 5-fold axis. The template strand enters from within the particle, and the dsRNA product of the initial replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.

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