Single%20Particle%20X-ray%20Diffraction

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Single Particle X-ray Diffraction - the Present
and the Future 
John Miao Stanford Synchrotron Radiation
Laboratory Stanford Linear Accelerator
Center   
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Nobel Prizes awarded to research related to the
phase problem F. Zernike (Physics in 1953),
for his invention of phase contrast method.
M. F. Perutz J. C. Kendrew (Chemistry in
1962), for their studies of the structures of
globular proteins. D. Gabor (Physics in 1971),
for his invention and development of the
holographic method. J. Karle H. Hauptman
(Chemistry in 1985) for their contributions to
the Direct Methods.
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A 200 ?m crystal (a 50 Å, 4 ? 104 unit cells)
Real
Reciprocal
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A 0.1 ?m crystal (a 50 Å, 20 unit cells)
Real
Reciprocal
F
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The Essence of the Oversampling Phasing
Method     Real Space ?F?
Reciprocal Space
Bragg-peak sampling

Oversampling
J. Miao, D. Sayre H. N. Chapman, J. Opt. Soc.
Am. A 15, 1662 (1998).
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The Oversampling Phasing Method
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An Iterative Algorithm
JJ. Fienup, Appl. Opt. 21, 2758 (1982). JJ.
Miao, J. Kirz D. Sayre, Acta Cryst. D 56, 1312
(2000).
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(a) A SEM image of a double-layered sample made
of Ni (2.7 x 2.5 x 1 ?m3)
(b) A coherent diffraction pattern from (a)
(the resolution at the edge is 8 nm)
(c) An image reconstructed from (b)
J. Miao et al., Phys. Rev. Lett. 89, 088303
(2002).
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The Reconstructed 3D structure
The reconstructed top pattern The reconstructed
bottom pattern
An iso-surface rendering of the reconstructed 3D
structure
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Direct determination of the absolute electron
density of nanostructured materials
I0 Measured by an X-ray photodiode I( )
Measured by a direct-illumination CCD
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(a) Coherent diffraction pattern from a porous
silica particle
(b) The reconstructed absolute electron density
(c) The absolute electron density distribution
within a 100 x 100 nm2 area
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Imaging Whole E. Coli Bacteria
(a) Light and fluorescence microscopy images of
E. Coli labeled with YFP and manganese oxide
(b) A Coherent X-ray diffraction pattern from E.
Coli
(c) An image reconstructed from (b).
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Radiation damage   SSolemn Baldwin, Science
218, 229-235 (1982).   Þ  ? With picosecond
pulse duration X-rays, biological specimens
remain morphological unchanged to an accuracy of
a few nm.     NNeutze, Wouts, Spoel, Weckert
Hajdu, Nature 400, 752-757 (2000). Þ ? With an
X-FEL of pulse leng. lt 50 fs and 3 x 1012 photons
focused down to a spot of 0.1 ?m, a 2D
diffraction pattern could be recorded from a
biomolecule before the radiation damage
manifests itself.  
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Orientation determination     Use the methods
developed in cryo-EM to determine the molecular
orientation based on many 2D diffraction
patterns.   Crowther, Phil. Trans. Roy. Soc.
Lond. B. 261, 221 (1971).   J. Frank, in
Three-Dimensional Electron Microscopy of
Macromolecular Assemblies, Academic Press
(1996).    Use laser fields to physically align
each molecule.   J. J. Larsen, K. N. Hald,
Bjerre, H. Stapelfeldt T. Seideman, Phys.
Rev. Lett. 85, 2470-2473 (2000).
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The 3D electron density map of a rubisco molecule
The active site of the molecule
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Procedures to Obtain Oversampled 3D Diffraction
Patterns

• Calculated oversampled 2D diffraction patterns
  from
• 106 identical molecules.
• Assumed that the orientation of each 2D
  diffraction pattern is known.
• Assembled an oversampled 3D diffraction pattern
  from these
• oversampled 2D diffraction patterns.
• (iv) Added Poisson noise to the 3D diffraction
  pattern.

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• One section of the oversampled 3D diffraction
• Pattern with RI 9.8 and 3x3x3 central pixels
  removed

(b) Top view of (a)
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The reconstructed 3D electron density map
The reconstructed active site
J. Miao, K. O. Hodgson D. Sayre, Proc. Natl.
Acad. Sci. USA 98, 6641 (2001).
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Reconstruction of the 3D diffraction pattern
obtained from 3 x 105 identical molecules with RI
16.6 and 3 x 3 x 3 central pixels removed.
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(a) The active site of the molecule from PDB
(b) The reconstruction with RI 9.8
(c) The reconstruction with RI 16.6
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• SSummary
• A new imaging methodology (i.e. single particle
  diffraction)
• has been developed by combining coherent X-rays
  with the oversampling method.
• The 2D and 3D imaging resolution of 8 nm and 50
  nm
• has been achieved.
• These results will pave a way for the
  development of atomic resolution 3D X-ray
  diffraction microscopy.
• In combination with the X-ray free electron
  lasers, single particle diffraction could be
  used to determine the 3D structure of single
  biomolcules at near atomic resolution.

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Acknowledgements

• B. Johnson K. Hodgson, Stanford Synchrotron
  Radiation Lab., Stanford University
• J. Kirz D. Sayre, SUNY at Stony Brook
• C. Larabell, UC San Francisco Lawrence Berkeley
  National Lab.
• M. LeGros, E. Anderson, Lawrence Berkeley
  National Lab.
• B. Lai, Advanced Photon Source, Argonne National
  Lab.
• T. Ishikawa, Y. Nishino, RIKEN/SPring-8, Japan
• J. Amonette, Pacific Northwest National Lab.