Application examples of single particle 3D reconstruction. Ning Gao Tsinghua University

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1 Application examples of single particle 3D reconstruction Ning Gao Tsinghua University

2 Electron Microscopes First electron microscope constructed by Ernst Ruska in 1930 s (1986 Nobel prize winner)

3 L. Marton, Electron Microscopy of Biological Objects. Nature, June 16, 1934

4 From 2D to 3D Arthur L. Robinson (1976) Electron Microscopy: Imaging Molecules in Three Dimensions. Science 192:

5 First 3D EM Structure Aaron Klug Nobel Prize,1982 DeRosier and Klug (1968) Reconstruction of Three Dimensional Structures from Electron Micrographs, Nature 217:130

6 First 3D structure of a Virus Tomato bushy stunt virus Crowther, Amos, Finch, DeRosier and Klug. (1970) Three Dimensional Reconstructions of Spherical Viruses by Fourier Synthesis from Electron Micrographs Nature 226:

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8 First 3D Structure of a Membrane Protein Electron Crystallography Henderson R, Unwin P. N. T. Three-dimensional model of purple membrane obtained by electron microscopy Nature 257: (1975)

9 How about objects without symmetry? EM reconstruction vs Crystallography Crystallization Crystallization in silico

10 omputer average of asymmetrical particles Joachim Frank Frank, Verschoor and Boublik (1981) Computer Averaging of Electron Micrographs of 40S Ribosomal Subunits. Science 214:

11 3D reconstruction of 50S subunit Tilt pairs M Radermacher, T Wagenknecht, A Verschoor, and J Frank (1987). Three-dimensional structure of the large ribosomal subunit from Escherichia coli. EMBO J Apr; 6(4):

12 Data collection geometries for 3D reconstruction

13 EM Sample preparation Water-free Negative staining technique, metal shadowing Chemical fixation, dehydration, substitution, plastic embedding Preserve water?

14 L. Marton, Electron Microscopy of Biological Objects. Nature, June 16, 1934

15 L. Marton, Electron Microscopy of Biological Objects. Nature, June 16, 1934

16 Cryo-freezing technique Robert M. Glaeser Key Taylor Jacques Dubochet Adrian et al., 1984, Cryo-electron microscopy of viruses, Nature 308:32-36

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18 Cryo-EM workflow

19 Cryo-EM: a versatile technique Single particles Asymmetric (ribosome) Symmetric (virus) 2D and tubular crystals Bacteriorhodopsin, Aquaporin 3D Microcrystals Filaments and fibers (Helical structure) Actin or tubulin filaments Whole cell or tissue sections

20 Blobology The ribosome of E. coli at 25 Å resolution Frank et al, 1995, Nature

21 Kühlbrandt, 2014, Science

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23 1.8-Å cryo-em map Cryo-EM Structure of apo-gdh Glutamate dehydrogenase, 334 Kd

24 Review on translation mrna Protein Decoding Peptide bond formatio

25 George E. Palade In 1974, Nobel Prize in Physiology or Medicine, for He added important methodological improvements both to the differential centrifugation and to the electron microscopy. In particular, he became instrumental in combing the two techniques... in order to obtain biologically basic information Palade, G.E., et al A small particulate component of the cytoplasm. J. Biophys. Biochem. Cytol. 1:59-68.

26 history of ribosome visualization Discovery of the ribosome by electron microscopy: George Palade (1955) EM of negatively stained ribosomes evidence of subunits: Hugh Huxley (1960) Location of ribosomal proteins, by immuno-electron microscopy: Jim Lake; Marina Stöffler-Meilicke, (1970s) Location of ribosomal proteins, by neutron scattering: Peter Moore (1980s) Exit site of polypeptide chain, by immuno-em: Jim Lake (1981) Exit tunnel of eukaryotic ribosomes, by electron crystallography of 2D crystals: Ron Milligan & Nigel Unwin (1986) Exit tunnel of prokaryotic ribosomes, by electron crystallography of 2D crystals: Ada Yonath (1987) mrna path, by immuno-em Schatzky (1991) Cryo-EM single-particle reconstruction of E. coli ribosome (1991, 1995): mrna path, exit tunnel, intersubunit bridges; trna (1996) and EF-G (1998) binding Joachim Frank X-ray structures of ribosomal subunits and ribosome (extremophiles): Tom Steitz & Peter Moore; Venki Ramakrishnan; Harry Noller; Ada Yonath ( ) Cryo-EM reveals ratchet rotation during translocation: Joachim Frank & Rajendra Agrawal (2000) Huxley and Zubay, 1960 Immuno-EM Neutron scattering Cryo-EM of 2D crystals Cryo-EM of single particles X-ray crystallography Courtesy of Joachim Frank

27 Static picture, at first The ribosome of E. coli at 25 Å resolution Frank et al, 1995, Nature

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29 Depict the architecture of the ribosome Frank, 2003

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31 Functional Cycle: elongation Initiation Elongation EF-Tu delivers aminoacylated trna to the A site Peptide formation Translocation Termination Recycling

32 translation

33 Peptide elongation cycle aa-trna >> accommodation peptidyl transfer >> translocation >> aa-trna >> accommodation

34 Visualization of Elongation Cycle kirromycin GDPNP fusidic acid (sordarin) GDPNP Frank, 2004

35 Let s take a look at translocation

36 Intersubunit motion Agarawal and Frank, 2001

37 Atomic details of intersubunit motion

38 Still not enough details! Keep in mind that during the t-rna translocation, the reading frame of the mrna must be maintained. More transient intermediate states are required to understand the underlying mechanism.

39 Uniqueness of 3DEM technique Multiple structures in one shot Scheres, 2007

40 Hybrid trna Agirrezabala et al, 2008, Mol Cell

41 Model for translocation Frank et al., 2007, PNAS

42 Aiming for more intermediate states? Sample preparation Hardware Microscope Detector Software Classification algorithm

43 trna intermediates revealed by ML3D Agirrezabala et al, 2012, PNAS

44 Four-dimensional cryo-em Cryo-EM technique could capture functional intermediates from a single dataset. Multiple conformers are present at the same time (multiple structures from a single shot) Data are collected at successive time points (time resolution)

45 Example: 4D cryo-em A, Ribosome complexes were prepared by adding a stoichiometric amount of deacylated trna fmet (green) that was cognate to the E-site codon, AUG, to ribosome complexes with fmetval-trna Val (purple) in the P site. Samples for cryo-em were taken before adding trna fmet, or after incubation for 1 min, 2 min, 5 min and 20 min at 37 C. b, Time course of retro-translocation determined from cryo-em images. Ribosome dynamics and trna movement by time-resolved electron cryomicroscopy. (2010). Fischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H. Nature. 466:

46 Ribosome dynamics and trna movement by time-resolved electron cryomicroscopy. (2010). Fischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H. Nature. 466:

47 Protein Translation Schmeing and Ramakrishnan 2009

48 70S ribosome (3.0 Å) unpublished

49 Visualization of rrna modification Fischer et al., Nature 520, (2015)

50 Mammalian Ribosome decoding center Shao et al., 2016, Cell 167:

51 Structural Snapshots of Actively Translating Human Ribosomes Behrmann et al., 2015, Cell

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53 2D morphological study 3D structural study Low resolution Atomic resolution Static structure Structural dynamics Qualitative Quantitative