Document Actions
X-Ray Serial and Single-Molecule Diffraction Imaging
Virtually all the known information about the molecular structure of biomolecules has been obtained by x-ray crystallography. Unfortunately, however, only 2% of the human proteome structures have yet been determined because of the extreme difficulty in producing high quality crystals. Worse yet, while most drugs target membrane proteins, these proteins cannot be crystallized.
It was suggested by Janos Hajdu (University of Uppsula) that an x-ray source of sufficient brightness and with a sufficiently short pulse could be used to produce a diffraction pattern using scattered x-rays from a single molecule before it is destroyed by the Coulomb explosion following rapid photoionization.1 The required brightness of ~1033 photons / (s ⋅ mm2 ⋅ mrad2 ⋅ (0.1% bandwidth)), x-ray energy of 5 keV, and pulse widths of 10 femtoseconds are expected to be achieved by the Linac Coherent Light Source (LCLS) 4th generation x-ray source funded by DOE and scheduled to be in operation by 2009.2 Successful implementation of this approach would represent a major breakthrough in structural biology and proteomics research.
Approach
To lay the scientific ground-work for single molecule diffraction measurements, CBST is supporting efforts by researchers at LLNL, UCD and LBNL to develop algorithms capable of reconstructing an object solely from diffraction data that can often be noisy and incomplete.
Another approach to single molecule imaging is also being pursued in a collaboration among Center researchers at LLNL, Arizona State University, and LBNL. Here, intense polarized laser light will be used to orient single biomolecules. This work will benefit the LCLS approach but is expected to also lead to a new near-term approach to crystal-less molecular structure measurement called serial crystallography.6, 7 Serial crystallography uses an established method to produce a stream of ice pellets, each containing an identical biomolecule (or alternatively a larger particle such as a single virus). The molecules will then be laser-aligned and allowed to pass through a synchrotron beam one-at-a-time. X-rays diffracted from the individual molecules will be collected and after contributions from ~104 molecules have accumulated, a diffraction pattern with sufficient signal-to-noise to make structure determinations will have been acquired. This method may lead to a new high-resolution method for obtaining structure information for proteins, protein complexes, and larger biological samples such as viral particles.
Systems/Experiments
Initial work has been conducted with model systems consisting of non-regular 3-dimensional arrangements of 50 nm-diameter gold spheres illuminated using 1.6 nm synchrotron radiation. New algorithms have been developed that for the first time are able to reconstruct high-resolution images directly from the recorded diffraction patterns.3 (See figure) The record setting resolutions (~10 nm) using this new approach of lenless imaging are not limited by the quality of the lens as in traditional approaches (ex. zone plate imaging4).
Accomplishments
CBST has been instrumental in advancing this approach by not only investing its own funds, but by encouraging substantial funding of other aspects of single molecule diffraction from LLNL, DOE, and SLAC. CBST co-hosted a large workshop at the Stanford Linear Accelerator Center to critically review the science and technology necessary to successfully image single biomolecules. The workshop resulted in a comprehensive DOE report on the feasibility and technical challenges of this project.5
Future Directions
Continued experiments and refinements of the diffraction imaging algorithms are planned including experiments at the TESLA Test Facility (TTF) short-pulse free-electron laser at the DESY synchrotron in Berlin.
The Center's goal for the above-described techniques is to revolutionize protein structure measurements by eliminating the need for crystals and to greatly improve x-ray imaging. Candidate molecules which will be among the first to be analyzed include clinically-important membrane proteins such as estrogen receptors, mammalian cytochrome P450, and tumor cell receptors (e.g., MUC-1 associated with breast cancer).
A collaborative effort with |
People
Henry Chapman, Janos Hajdu
Stefano Marchesini
A. Barty
B. Segelke
S.P. Hau-Riege
Richard London
A. Szöke
Eugene Ingerman
David Shapiro
C. Cui
H. He
Malcolm Howells
Bruce Doak
Uwe Weierstal
John Spence
References
1. Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752-757 (2000).
2. http://www-ssrl.slac.stanford.edu/lcls
3. Hau-Riege, S.P. et al. SPEDEN: reconstructing single particles from their diffraction patterns. Acta Crystallogr A 60, 294-305 (2004).
4. Chao, W., Harteneck, B.D., Liddle, J.A., Anderson, E.H. & Attwood, D.T. Soft X-ray microscopy at a spatial resolution better than 15 nm. Nature 435, 1210-1213 (2005).
5. http://renewal.cbst.ucdavis.edu/sci/LCLS_DOE.pdf
6. Spence, J.C. & Doak, R.B. Single molecule diffraction. Phys Rev Lett 92, 198102 (2004).
7. Spence, J.C. et al. Diffraction and imaging from a beam of laser-aligned proteins: resolution limits. Acta Crystallogr A 61, 237-245 (2005).