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6 Publications

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    Grigorieff Lab
    11/11/11 | Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy.
    Henderson R, Chen S, Chen JZ, Grigorieff N, Passmore LA, Ciccarelli L, Rubinstein JL, Crowther RA, Stewart PL, Rosenthal PB
    Journal of Molecular Biology. 2011 Nov 11;413(5):1028-46. doi: 10.1016/j.jmb.2011.09.008

    The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass. The angular uncertainty is less than 1° for particles of high molecular mass ( 50 MDa), several degrees for particles in the range 1-5 MDa, and tens of degrees for particles below 1 MDa. Orientational uncertainty may be the major contributor to the effective temperature factor (B-factor) describing contrast loss and therefore the maximum resolution of a structure determination. We also made two unexpected observations. Single particles that are known to be flexible showed a wider spread in orientation accuracy, and the orientations of the largest particles examined changed by several degrees during typical low-dose exposures. Smaller particles presumably also reorient during the exposure; hence, specimen movement is a second major factor that limits resolution. Tilt pairs thus enable assessment of orientation accuracy, map quality, specimen motion, and conformational heterogeneity. A convincing tilt-pair parameter plot, where 60% of the particles show a single cluster around the expected tilt axis and tilt angle, provides confidence in a structure determined using electron cryomicroscopy.

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    Grigorieff Lab
    10/01/11 | An adaptation of the Wiener filter suitable for analyzing images of isolated single particles.
    Sindelar CV, Grigorieff N
    Journal of Structural Biology. 2011 Oct;176(1):60-74. doi: 10.1016/j.jsb.2011.06.010

    The Wiener filter is a standard means of optimizing the signal in sums of aligned, noisy images obtained by electron cryo-microscopy (cryo-EM). However, estimation of the resolution-dependent ("spectral") signal-to-noise ratio (SSNR) from the input data has remained problematic, and error reduction due to specific application of the SSNR term within a Wiener filter has not been reported. Here we describe an adjustment to the Wiener filter for optimal summation of images of isolated particles surrounded by large regions of featureless background, as is typically the case in single-particle cryo-EM applications. We show that the density within the particle area can be optimized, in the least-squares sense, by scaling the SSNR term found in the conventional Wiener filter by a factor that reflects the fraction of the image field occupied by the particle. We also give related expressions that allow the SSNR to be computed for application in this new filter, by incorporating a masking step into a Fourier Ring Correlation (FRC), a standard resolution measure. Furthermore, we show that this masked FRC estimation scheme substantially improves on the accuracy of conventional SSNR estimation methods. We demonstrate the validity of our new approach in numeric tests with simulated data corresponding to realistic cryo-EM imaging conditions. This variation of the Wiener filter and accompanying derivation should prove useful for a variety of single-particle cryo-EM applications, including 3D reconstruction.

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    Grigorieff Lab
    09/21/11 | Structural complexity of a composite amyloid fibril.
    Lewandowski JR, van der Wel PC, Rigney M, Grigorieff N, Griffin RG
    Journal of the American Chemical Society. 2011 Sep 21;133(37):14686-98. doi: 10.1021/ja203736z

    The molecular structure of amyloid fibrils and the mechanism of their formation are of substantial medical and biological importance, but present an ongoing experimental and computational challenge. An early high-resolution view of amyloid-like structure was obtained on amyloid-like crystals of a small fragment of the yeast prion protein Sup35p: the peptide GNNQQNY. As GNNQQNY also forms amyloid-like fibrils under similar conditions, it has been theorized that the crystal’s structural features are shared by the fibrils. Here we apply magic-angle-spinning (MAS) NMR to examine the structure and dynamics of these fibrils. Previously multiple NMR signals were observed for such samples, seemingly consistent with the presence of polymorphic fibrils. Here we demonstrate that peptides with these three distinct conformations instead assemble together into composite protofilaments. Electron microscopy (EM) of the ribbon-like fibrils indicates that these protofilaments combine in differing ways to form striations of variable widths, presenting another level of structural complexity. Structural and dynamic NMR data reveal the presence of highly restricted side-chain conformations involved in interfaces between differently structured peptides, likely comprising interdigitated steric zippers. We outline molecular interfaces that are consistent with the observed EM and NMR data. The rigid and uniform structure of the GNNQQNY crystals is found to contrast distinctly with the more complex structural and dynamic nature of these "composite" amyloid fibrils. These results provide insight into the fibril-crystal distinction and also indicate a necessary caution with respect to the extrapolation of crystal structures to the study of fibril structure and formation.

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    Grigorieff Lab
    06/01/11 | Recent progress in understanding Alzheimer’s β-amyloid structures.
    Fändrich M, Schmidt M, Grigorieff N
    Trends in Biochemical Sciences. 2011 Jun;36(6):338-45. doi: 10.1016/j.tibs.2011.02.002

    The formation of amyloid fibrils, protofibrils and oligomers from the β-amyloid (Aβ) peptide represents a hallmark of Alzheimer’s disease. Aβ-peptide-derived assemblies might be crucial for disease onset, but determining their atomic structures has proven to be a major challenge. Progress over the past 5 years has yielded substantial new data obtained with improved methodologies including electron cryo-microscopy and NMR. It is now possible to resolve the global fibril topology and the cross-β sheet organization within protofilaments, and to identify residues that are crucial for stabilizing secondary structural elements and peptide conformations within specific assemblies. These data have significantly enhanced our understanding of the mechanism of Aβ aggregation and have illuminated the possible relevance of specific conformers for neurodegenerative pathologies.

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    Grigorieff Lab
    04/01/11 | Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy.
    Grigorieff N, Harrison SC
    Current Opinion in Structural Biology. 2011 Apr;21(2):265-73. doi: 10.1016/j.sbi.2011.01.008

    Nine different near-atomic resolution structures of icosahedral viruses, determined by electron cryo-microscopy and published between early 2008 and late 2010, fulfil predictions made 15 years ago that single-particle cryo-EM techniques could visualize molecular detail at 3-4A resolution. This review summarizes technical developments, both in instrumentation and in computation, that have led to the new structures, which advance our understanding of virus assembly and cell entry.

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    Grigorieff Lab
    01/19/11 | Atomic model of an infectious rotavirus particle.
    Settembre EC, Chen JZ, Dormitzer PR, Grigorieff N, Harrison SC
    The EMBO Journal. 2011 Jan 19;30(2):408-16. doi: 10.1038/emboj.2010.322

    Non-enveloped viruses of different types have evolved distinct mechanisms for penetrating a cellular membrane during infection. Rotavirus penetration appears to occur by a process resembling enveloped-virus fusion: membrane distortion linked to conformational changes in a viral protein. Evidence for such a mechanism comes from crystallographic analyses of fragments of VP4, the rotavirus-penetration protein, and infectivity analyses of structure-based VP4 mutants. We describe here the structure of an infectious rotavirus particle determined by electron cryomicroscopy (cryoEM) and single-particle analysis at about 4.3 Å resolution. The cryoEM image reconstruction permits a nearly complete trace of the VP4 polypeptide chain, including the positions of most side chains. It shows how the two subfragments of VP4 (VP8(*) and VP5(*)) retain their association after proteolytic cleavage, reveals multiple structural roles for the β-barrel domain of VP5(*), and specifies interactions of VP4 with other capsid proteins. The virion model allows us to integrate structural and functional information into a coherent mechanism for rotavirus entry.

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