This program has two main tasks: production of nano-fabricated and nano-arrayed samples; and measurement of diffraction data from the various samples to be investigated during the life of this Centre. Ancillary responsibility includes provision of parameters and feedback into the Theory and Modelling Program, such as measurement of the input curved beam parameters and investigation of partial coherence effects.
The Centre aims to develop new methods for macromolecular structure determination based on "single molecule" diffraction. While this is the ultimate aim and may be realizable with the advent of X-FELs (such as the TESLA project in Hamburg or the LCLS at Stanford), there is a range of intermediate approaches that seem attractive and realizable with 3rd generation conventional synchrotron sources including:
- initial tests involving high atomic weight nanostructured arrays and individual complexes - the high atomic weight increases diffractive power and thus increases signal to noise
- the imaging of cellular and sub-cellular structures as described in the Biological Sciences Program - these will initially incorporate heavy atoms into the structures. This work will be in parallel with imaging under the Short-Wavelength Laser Program.
- imaging of nano-arrayed proteins, stained then unstained and culminating in the structural determination of macromolecular complexes described in the Biological Sciences Program - these molecules would preferably be oriented about all three axes, but even order with respect to two rotational axes would likely be a good start and reduce the diffuse nature of the diffraction pattern from 3- to 1-dimension and greatly increase expected signal to noise compared to actual single molecule data collection.
Sample development: Cellular and macromolecular sample preparation is described in the Biological Sciences Program. In this Program, we will use a nano-fabrication method that is based around direct writing onto a surface using a coated AFM tip. Thus chemical treatment of a surface at precisely defined spatial coordinates can allow for etching and further treatment of that surface to produce nano-scale gold arrays. Alternatively, small clusters of protein molecules can be directly deposited onto a suitable surface. These methods for fabrication of artificial structures are now becoming well-established , , and we will develop an in-house capacity for the nano-engineering of suitable test samples. This will build on existing experience in AFM techniques developed in the School of Physics at the University of Melbourne as well as on the microfabrication experience of CI Peele. Another approach for making artificially produced arrays is by surface templating of a substrate followed by attachment of the molecules to the substrate. Through work already underway with PI Wilkins we will have access to key infrastructure equipment for producing suitable templated surfaces (e.g. e-beam lithography) and also to a considerable reservoir of expertise in the molecular structuring of surfaces for a wide range of applications. That expertise will also be tapped to develop the capacity to provide even smaller features via e-beam lithography and at the nanofabrication facility to be commissioned under CI Peele.
X-ray experiments: The experimental work will be performed in collaboration with our partners at the SPring8 synchrotron in Japan and at the Advanced Photon Source synchrotron in the USA via the Australian Synchrotron Research Program. The information obtained will be fed into both the Theory and Modelling Program and the Beamline Development Program. The Centre possesses a vast reservoir of experience in synchrotron, crystallographic and SAXS measurement methods in the persons of its CIs and PIs. Essentially we will be combining existing phase imaging methods (Nugent, Peele, McNulty) with cryogenic sample handling and low dosage methods as described in the Biological Sciences Program (Varghese), with SAXS methods (Lewis, Berry, Wilkins) and with higher angle coherent diffractive methods (Ishikawa, Miao, Chapman).
The experiments corresponding to the samples described above represent a series of intermediate steps towards the goal of macromolecular structural determination. An alternative intermediate step is to relax the resolution goal. Collection of diffraction data from protein samples by grazing angle small angle x-ray scattering could give sufficient signal to provide information on the shape of the macromolecules, while large-angle X-ray scattering methods (albeit with weaker signal) would give high-resolution structural information. This is intended to open up a major new approach to macromolecular structure determination. Initial characterization of such samples would be carried out using a very high-performance laboratory SAXS instrument that is currently being installed at CSIRO in Clayton as part of a Monash-CSIRO collaboration.
Critical issues in achieving success in all of these investigations lie in the quality and density of 2-D structures that can be produced, questions of minimization of radiation damage to bio samples, e.g. by use of cryo techniques and the acquisition or development of very high signal to noise (i.e. photon counting) X-ray detectors. The fact that 2-D surface diffraction using X-rays is a well-established and successful technique for inorganic samples gives us grounds for confidence that these techniques may be extended to biological materials, even though there are clearly many additional hurdles to be overcome.
In electron diffraction it is possible to reconstruct single molecules by summing the signal from many individual images via sophisticated pattern recognition algorithms. A similar approach might be envisaged here, where a two-dimensional crystal can be constructed and the focussed beam of the x-ray source scanned across it. If the incident beam is well-characterised - and this program will develop methods for that -the diffracted beam will then be able to be added from each beam position and the structure recovered. This Program, in conjunction with the Theory and Modelling Program, will develop the modelling and experimental tools to allow molecular reconstruction in this, or a related, manner. This Program will work with the Biological Sciences Program and with the Short-Wavelength Laser Program, particularly in relation to cellular and sub-cellular imaging to test and prove these ideas. In this work we will draw closely upon our international partners working on the Linac Coherent Light Source X-FEL.
The result of this program will be a deep appreciation of the experimental aspects of a molecular scale diffraction experiment and the development of a detailed experimental design for the structural determination of a membrane protein.
