Single Particle X-ray Diffraction
- the Present and the Future
John Miao
Stanford Synchrotron Radiation Laboratory
Stanford Linear Accelerator Center
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”.
A 200 m crystal (a = 50 Å, 4  104 unit cells)
Real

Reciprocal
|ℱ

|


A 0.1 m crystal (a = 50 Å, 20 unit cells)
Real

Reciprocal
|ℱ
|


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).
The Oversampling Phasing Method
An Iterative Algorithm
JJ. Fienup, Appl. Opt. 21, 2758 (1982).
JJ. Miao, J. Kirz & D. Sayre, Acta Cryst. D 56, 1312 (2000).
(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).
The Reconstructed 3D structure
The reconstructed top pattern
The reconstructed bottom pattern
An iso-surface rendering of the reconstructed 3D structure
Direct determination of the absolute electron
density of nanostructured materials
 I P r2
 2
I (k )  0 2 e F (k )
r
I0: Measured by an X-ray photodiode

I(k): Measured by a direct-illumination CCD
(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
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).
Radiation damage
SSolemn & Baldwin, Science 218, 229-235 (1982).
  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).

With an X-FEL of pulse leng. < 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.
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).
The 3D electron density map of a rubisco molecule
The active site of the molecule
Procedures to Obtain Oversampled 3D Diffraction Patterns
(i) Calculated oversampled 2D diffraction patterns from
106 identical molecules.
(ii) Assumed that the orientation of each 2D diffraction pattern is known.
(ii) Assembled an oversampled 3D diffraction pattern from these
oversampled 2D diffraction patterns.
(iv) Added Poisson noise to the 3D diffraction pattern.
RI 
I
k x ,k y ,k z
calculated
(k x , k y , k z )  I noisy (k x , k y , k z )
I
k x ,k y ,k z
calculated
(k x , k y , k z )
(a) One section of the oversampled 3D diffraction
Pattern with RI = 9.8% and 3x3x3 central pixels removed
(b) Top view of (a)
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).
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.
(a) The active site of the
(b) The reconstruction
(c) The reconstruction
molecule from PDB
with RI = 9.8%
with RI = 16.6%
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.
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.