Single Particle X-ray Diffraction- the Present and the Future 

John MiaoStanford 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)

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Real Reciprocal

A 0.1 m crystal (a = 50 Å, 20 unit cells)

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Real Reciprocal

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

2

2

20 )()( kFr

rPIkI e

I0: Measured by an X-ray photodiode

I( ): Measured by a direct-illumination CCDk

(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

(b) A Coherent X-ray diffraction pattern

from E. Coli

(c) An image reconstructed from (b).

(a) Light and fluorescence microscopy images

of E. Coli labeled with YFP and manganese oxide

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.

zyx

zyx

kkkzyxcalculated

kkkzyxnoisyzyxcalculated

IkkkI

kkkIkkkI

R

,,

,,

),,(

),,(),,(

(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

molecule from PDB

(b) The reconstruction

with RI = 9.8%

(c) The reconstruction

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.