John Briggs - EMBL HamburgJohn Briggs Subject EMBO Course on Methods for Biological Macromolecules...
Transcript of John Briggs - EMBL HamburgJohn Briggs Subject EMBO Course on Methods for Biological Macromolecules...
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John Briggs
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Structure across range of resolutions and samples
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Easier than crystallography?
Yeast mitochondrial ribosome Amunts et al. Science 2014
Membrane Proteins
TRPV1 ion channel Liao et al Nature 2013
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Solve structure “in situ”
B. D. Engel, M. Schaffer, S. Albert, S. Asano, J. M. Plitzko and W. Baumeister: In situ structural analysis of Golgi intracisternal protein arrays. Proceedings of the National Academy of Sciences USA, September 8, 2015
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Mixed samples, populations
N Fischer et al. Nature 2010
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1. Preparation methods which preserve 3D structure 2. A microscope which allows us to image the sample 3. Different views of the object 4. Computational approaches for producing 3D reconstructions from projections 5. Approaches for assessing our 3D reconstruction and interpreting it in terms of biology
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Requirements for sample preparation: 3D structure preserved at appropriate resolution Sample thin enough for electron beam preparation Sample stable in a vacuum
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1. Negative staining
2. Vitrification (for cryo-EM)
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Bettina Boettcher
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Micrograph of negatively stained sample
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3 mm
Bridget Carragher
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Movie from the Baumeister lab, Max-Planck Institute for Biochemistry
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It is a projection image
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It is a noisy projection image
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Slide: Andy Hoenger
It is noisy because of limited electron dose
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Limited exposure
High-resolution information is lost as exposure accumulates
elifesciences.orgTOOLS AND RESOURCES
Measuring the optimal exposure for singleparticle cryo-EM using a 2.6 Areconstruction of rotavirus VP6Timothy Grant1, Nikolaus Grigorieff1,2*
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States;2Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center,Brandeis University, Waltham, United States
Abstract Biological specimens suffer radiation damage when imaged in an electron microscope,ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can bedefined that maximizes the signal-to-noise ratio in the image. Using a 2.6 A resolution single particlecryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of100 electrons/A2, we obtained accurate measurements of optimal exposure values over a wide rangeof resolutions. At low and intermediate resolutions, our measured values are considerably higherthan obtained previously for crystalline specimens, indicating that both images and movies should becollected with higher exposures than are generally used. We demonstrate a method of using ouroptimal exposure values to filter movie frames, yielding images with improved contrast that lead tohigher resolution reconstructions. This ‘high-exposure’ technique should benefit cryo-EM work on alltypes of samples, especially those of relatively low-molecular mass.DOI: 10.7554/eLife.06980.001
IntroductionElectron microscopy of isolated macromolecules and their complexes (single particles) embeddedin a thin layer of vitreous ice (cryo-EM) has recently led to a number of structures determined atnear-atomic resolution (Liao et al., 2013; Allegretti et al., 2014; Bartesaghi et al., 2014; Wonget al., 2014), a level of detail that had previously been restricted to X-ray crystallography andNMR (for a recent review of the technique, see Cheng et al., 2015). A limiting factor in theresolution of EM images of biological specimens is radiation damage because the imaging of suchspecimens ultimately relies on the interaction of electrons with the sample. Some of theseinteractions will result in energy being deposited in the specimen, and these will cause radiationdamage (Glaeser, 1971; Henderson, 1995). The radiation damage fundamentally limits theinformation present in the images because the useful signal added per incident electron decreaseswith increasing electron exposure, while the added noise (image contrast originating from otherparts of the sample as well as inelastic scattering) remains approximately constant. If the signalgain per unit exposure is known at a given resolution, an optimal exposure can be chosen thatwill maximize the signal-to-noise ratio (SNR) at that resolution (Hayward and Glaeser, 1979;Baker et al., 2010).
The rate of exposure-dependent signal decay has been measured by following the intensities ofthe fading diffraction spots in exposure series obtained from 2D and thin 3D crystals (Unwin andHenderson, 1975; Hayward and Glaeser, 1979; Stark et al., 1996; Baker et al., 2010). Thesestudies demonstrated that higher resolution intensities tend to fade faster than lower resolutionintensities, and that the rate of fading for all resolutions is slowed under liquid nitrogen conditionsrelative to room temperature conditions. The fading of the spots can be described by an exponential
*For correspondence: niko@
grigorieff.org
Competing interests: The
authors declare that no
competing interests exist.
Funding: See page 17
Received: 12 February 2015
Accepted: 28 May 2015
Published: 29 May 2015
Reviewing editor: Wesley I
Sundquist, University of Utah
School of Medicine, United
States
Copyright Grant and
Grigorieff. This article is
distributed under the terms of
the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Grant and Grigorieff. eLife 2015;4:e06980. DOI: 10.7554/eLife.06980 1 of 19
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It is a noisy projection image
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It is a noisy projection image modulated by a transfer function
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How to go from this to a 3D reconstruction?
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Key concept
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Single particle EM The particles are randomly oriented. Each image of the particle gives us a different view
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Key concept
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Slide: Andy Hoenger
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Slide: Bettina Boettcher
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Movie from the Baumeister lab, Max-Planck Institute for Biochemistry
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Computational tools for producing a 3D reconstruction from projections CTF correction Filtering Alignment Classification Averaging Angle assignment 3D reconstruction Refinement Sharpening/weighting
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Find the particles within the image
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Extract the particles from the image
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Slide: Andy Hoenger
Averaging is going to require alignment…
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Align the particles – rotationally and translationally
Bettina Boettcher
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Classify and average similar images: Example: F-type ATP-Synthase, Bettina Boettcher
Bettina Boettcher
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Classify and average similar images: Example: F-type ATP-Synthase, Bettina Boettcher And then use to improve the alignment… iteration
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Angle assignment How to these views all relate to each other?
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Slide: Bettina Boettcher
Angle assignment
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If you have a starting model: Projecting a model and compare images to the projections (projection matching)
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Next – generate the 3D reconstruction In real space or in Fourier space.
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3D reconstruction by back projection
A. Frangakis
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3D reconstruction by back projection
A. Frangakis
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Once we have a model, we can compare new images to the model to work out their orientation… and refine our structure to high resolution. (projection matching)
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Nogales E , Grigorieff N J Cell Biol 2001;152:F1-F10
© 2001 The Rockefeller University Press
Iterate…
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Barford lab Subramaniam lab
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A.Hoenger
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Tamir Gonen, Janelia/HHMI
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In situ or in vitro?
Simple systems Purified components in vitro
Complex systems Components in situ
Low-resolution “blobs” High-resolution detail
Cryo-electron tomography Single-particle cryo-EM
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Movie from the Baumeister lab, Max-Planck Institute for Biochemistry
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Julia Mahamid et al. Science 2016;351:969-972
Published by AAAS
Ribosomes at 28 Å resolution
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Subtomogram averaging
tomogram
subtomograms(randomly oriented)
aligned toreference subtomograms
(aligned)
averaged subtomograms(new reference)
use new reference for alignmentiterate until reference is stable
Figure 1
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N Fischer et al. Nature 2010
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Solve multiple conformations from same sample
Abeyrathna et al. eLife 2016;5:e14874
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Solve multiple conformations from same sample
Bai et al. eLife 2015;4:e11182
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(A) Three views of a cryo-EM map of the 80S ribosome from yeast (32), with arrows indicating four key conformational changes associated with the elongation work cycle of the ribosome.
Ali Dashti et al. PNAS 2014;111:17492-17497
©2014 by National Academy of Sciences
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Ali Dashti et al. PNAS 2014;111:17492-17497
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Cryo-EM can be used to: Determine structures at a wide range of resolution Determine structures within complex environments Determine multiple structures from mixed samples Determine conformational landscape of proteins, or energy landscape of proteins.