Single-Particle Reconstruction Story in a Sample Joachim Frank - PowerPoint PPT Presentation

Single-Particle Reconstruction Story in a Sample Joachim Frank Department of Biochemistry and Molecular Biophysics and Department of Biological Sciences Columbia University

12/08/2017 Nobel Lecture, Stockholm 2

Drawing by Graham Colm, courtesy of Wikimedia Commons

Archives of the Max Planck Society, Berlin Walter Hoppe (March 21, 1917 – November 3, 1986) 12/08/2017 Nobel Lecture, Stockholm 4

TO CAPTURE A THREE-DIMENSIONAL OBJECT . . . . . . MULTIPLE VIEWS ARE NEEDED 12/08/2017 Nobel Lecture, Stockholm 5

THREE-DIMENSIONAL RECONSTRUCTION: HELICAL SYMMETRY Photo credit Brian Clark Aaron Klug David DeRosier 12/08/2017 Nobel Lecture, Stockholm 6

THREE-DIMENSIONAL RECONSTRUCTION: VIRUSES WITH ICOSAHEDRAL SYMMETRY R. A. Crowther, Phil. Trans. Roy. Soc. 1971 Tony Crowther 12/08/2017 Nobel Lecture, Stockholm 7

Alpbach, site of workshops on protein X-ray crystallography organized by Walter Hoppe and Max Perutz 12/08/2017 Nobel Lecture, Stockholm 8

THREE-DIMENSIONAL RECONSTRUCTION: TWO-DIMENSIONAL CRYSTAL (PURPLE MEMBRANE PROTEIN) Henderson and Unwin, Nature 1975 12/08/2017 Nobel Lecture, Stockholm 9

Molecule captured in different views Three-dimensional reconstruction produces a 3D image, or density map Sali et al. Nature 2003 12/08/2017 Nobel Lecture, Stockholm 10

SINGLE-PARTICLE PROJECTIONS – MOLECULES NEGATIVELY STAINED 12/08/2017 Nobel Lecture, Stockholm 11

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• ALIGN & AVERAGE • ESTIMATE RESOLUTION • SORT/CLASSIFY • FIND ANGLES • RECONSTRUCT 12/08/2017 Nobel Lecture, Stockholm 13

Peak Cross-correlation function Autocorrelation function J. Frank, Ph.D. thesis 1970 12/08/2017 Nobel Lecture, Stockholm 14

SIMULTANEOUS SHIFT AND ROTATION ALIGNMENT 12/08/2017 Nobel Lecture, Stockholm 15

CONDITIONS FOR ALIGNMENT OF TWO IMAGES OF A MOLECULE OF SIZE D 3 ≥ D 2 c dp crit PARTICLE SIZE > 3/[CONTRAST 2 x RESOLUTION (in Å) x CRITICAL ELECTRON DOSE] Saxton & Frank, Ultramicroscopy 1977 12/08/2017 Nobel Lecture, Stockholm 16

GLUTAMINE SYNTHETASE 12/08/2017 Nobel Lecture, Stockholm 17

ACETYLCHOLINE RECEPTOR Zingsheim et al., Proc. Natl. Adad. Sci. USA 1980 12/08/2017 Nobel Lecture, Stockholm 18

40S RIBOSOMAL SUBUNIT FROM HELA CELLS Frank et al. Science 1981 12/08/2017 Nobel Lecture, Stockholm 19

RESOLUTION = EXTENT OF REPRODUCIBILITY IN FOURIER SPACE BY OPTICAL DIFFRACTION J. Frank, Ph.D. thesis 1970 12/08/2017 Nobel Lecture, Stockholm 20

RESOLUTION = EXTENT OF REPRODUCIBILITY IN FOURIER SPACE USING THE COMPUTER Differential Phase Residual 45° J. Frank et al., Science 1981 12/08/2017 Nobel Lecture, Stockholm 21

Limulus polyphemus hemocyanin Van Heel and Frank, Ultramicroscopy 1981 12/08/2017 Nobel Lecture, Stockholm 22

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12/08/2017 Nobel Lecture, Stockholm 24

Jet Propulsion Laboratory, Pasadena, California 12/08/2017 Nobel Lecture, Stockholm 25

RANDOM-CONICAL RECONSTRUCTION – PRINCIPLE J. Frank, overhead 1979 12/08/2017 Nobel Lecture, Stockholm 26

RANDOM-CONICAL RECONSTRUCTION – PRINCIPLE (FANCY VERSION) J. Frank, American Scientist 1998 12/08/2017 Nobel Lecture, Stockholm 27

RECONSTRUCTION OF 50S RIBOSOMAL SUBUNIT FROM E. COLI RIBOSOME Radermacher et al., EMBO J. 1987 12/08/2017 Nobel Lecture, Stockholm 28

SINGLE-PARTICLE PROJECTIONS – MOLECULES EMBEDDED IN ICE 12/08/2017 Nobel Lecture, Stockholm 29

CONTRAST TRANSFER FUNCTION CORRECTION FROM DEFOCUS SERIES * N SNR k H ( ) ( ) k = ∑ = n n F ( ) W k F ( ) ( ) W k ( ) k k n n n N ∑ + = 2 1 n ( ) | ( ) | 1 SNR k H k n n = Penczek et al., Scanning Microscopy 1997 n 1 12/08/2017 Nobel Lecture, Stockholm 30

E. coli ribosome Octopus hemocyanin Calcium Release Channel Frank et al., Nature 1995 Lambert et al., 1994 Radermacher et al., 1994 12/08/2017 Nobel Lecture, Stockholm 31

CRYO-EM RECONSTRUCTION ASSISTING IN PHASING THE X-RAY STRUCTURE Ban et al., Cell 1998 12/08/2017 Nobel Lecture, Stockholm 32

ANGULAR REFINEMENT (3D PROJECTION MATCHING) J. Frank, Molecular Machines in Biology, Ch. 2, Cambridge University Press 2011 12/08/2017 Nobel Lecture, Stockholm 33

RATCHET-LIKE INTERSUBUNIT MOTION Frank & Agrawal Nature 2000 Valle et al., Cell 2003 12/08/2017 Nobel Lecture, Stockholm 34

tRNA AS A MOLECULAR SPRING DURING DECODING Valle et al., Nat. Struct. Biol. 2003 12/08/2017 Nobel Lecture, Stockholm 35

HEPATITIS C VIRUS IRES INVADING THE HOST RIBOSOME Spahn et al., Science 2001 12/08/2017 Nobel Lecture, Stockholm 36

ATOMIC MODELS THROUGH FLEXIBLE FITTING I Villa et al., PNAS 2009 12/08/2017 Nobel Lecture, Stockholm 37

ATOMIC MODELS THROUGH FLEXIBLE FITTING II Hashem et al., Nature 2013 12/08/2017 Nobel Lecture, Stockholm 38

Top: classes derived by supervised classification Bottom: classes derived by Maximum Likelihood classification 11,415 particles in common Scheres et al., Nature Methods 2007 12/08/2017 Nobel Lecture, Stockholm 39

“STORY IN A SAMPLE” EF-G mutant binding to “PRE” complex Sun et al., Nucl. Acid Res. 2015 Adapted from Li et al., Science Adv. 2015 12/08/2017 Nobel Lecture, Stockholm 40

T. cruzi ribosome Calcium release channel AMPA receptor Liu et al., Des Georges et al., Twomey et al., PNAS 2016 Cell 2017 Nature 2017 12/08/2017 Nobel Lecture, Stockholm 41

Wadsworth Center, Albany, New York State Department of Health Department of Biomedical Sciences, University at Albany Since 2008: Department of Biochemistry and Molecular Biophysics, and Department of Biological Sciences, Columbia University Funding: National Institute of General Medical Science, NIH National Science Foundation Howard Hughes Medical Institute 12/08/2017 Nobel Lecture, Stockholm 42