The Macromolecular Characterisation beamline proposal is for a bending magnet beamline in which circular dichroism (CD) and small-angle x-ray scattering (SAXS) will be measured simultaneously on the same sample. In the context of biological macromolecules, SAXS and CD are highly synergistic. SAXS is sensitive to the tertiary structure of a molecule (its over-all shape), while CD measures the secondary structure (the way the polymer chains are arranged). Together the techniques cover the two main aspects of macromolecular structure. The beamline will largely be aimed at the life science research community including biochemists, protein chemists and structural biologists and will meet a growing need for structural characterisation of soluble proteins.

The beamline will consist of three integrated components, a SAXS instrument, a CD instrument and software and hardware for automating sample handling and data collection, reduction and analysis.

The SAXS component of the beamline is being developed by Nigel Kirby who designed, built and runs the Australian Synchrotron SAXS beamline. The x-ray optics will consist of a 27 meter beamline with a multilayer monochromator positioned 14 meters from the sample. A set of Kirkpatric-Baez mirrors between the monochromator and the sample will serve to steer the beam to the sample with minimal focusing. The instrument will deliver 2x1012 photons per second to the sample position and with a fixed 1.6 meter camera length and a Pilatus 2M detector (Dectris) this setup will allow collection of SAXS data in a Q range of 0.01 to 0.5 Å-1 with exposure times of between 1 and 10 seconds. The x-ray beam will have a spot size of 200x200 um (full-width half-max) at the sample. By optimising the beamline specifically for measuring biological macromolecules it is anticipated that the beamline will perform comparably to the existing SAXS beamline for static measurement of proteins in the size range of 1-100 KDa without need for an insertion device. Beamline 12.3.1 at the Advanced Light Source is an example of a successful bending magnet biological SAXS beamline.

The CD component of the beamline is based on existing CD beamlines at the SRS, Diamond and Soleil synchrotrons. The bending magnet source will produce a fan of white light with a divergence of 170 mrad (ten degrees). The SAXS monochromator will utilise approximately 6 mrad of the centre of this fan of radiation. A mirror positioned close to the radiation source will deflect 30-40 mrad of white light upwards and outwards to the UV monochromator that will be positioned above the x-ray optics. The monochromator will house optics to propagate either monochromatic or pink beam to measure CD in either scanning or energy dispersive modes respectively. The UV beam will be angled down towards the sample with vertical and horizontal focusing mirrors assisting with steering and focus . The UV beam will cross the sample at an angle and have a spot of 300-500 um at the sample position. The photomultiplier or photoanalyser array will be mounted below the nose cone of the SAXS camera.

The sample environment and automation is an important part of the beamline design. The sample stage will allow for temperature control and for samples to be measured in a static or flow configuration. Customised cuvettes with multiple windows will be designed to facilitate measurement of SAXS and CD from the same sample while taking into account the techniques different requirements for pathlength and window materials. The endstation will also be designed such that traditional CD and SAXS sample holders can be accommodated.

The beamline will include an integrated high-pressure liquid chromatography (HPLC) system that will be used to deliver the samples, this will include an auto-sample loader allowing for a remote access mode of data collection. Via the HPLC, samples will go through a final purification step directly before analysis and will be characterised prior to measurement. The HPLC will load a sample from a 96 well plate automated sample loader to an analytical size exclusion column. This will separate the sample from aggregate and give an elution profile.

The sample will go through absorbance and refractive index measurement to give an accurate concentration. An inline multi-angle laser scattering instrument will characterise monodispersity, molecular weight and will make a measurement of radius of gyration before the sample is delivered into the SAXS and CD sample environment. Measuring sample concentration and monodispersity in-line with SAXS and CD will allow the data to be converted automatically to normalised units and compared with calibration standards for validation. This will reduce uncertainties of data quality that are implicit in SAXS and CD techniques by providing cross-validation.

Producing validated and normalised SAXS and CD data will allow automation of data analysis to an extent that has not previously been possible. Data collection software will be developed to allow easy selection of the many components and options of the beamline and also to harvest metadata surrounding the sample and experiment and interface with automated downstream data analysis. This will include on-the-fly secondary structure calculations from CD spectra and de novo modeling of protein structures from the SAXS data. Furthermore, there is an opportunity to truly integrate the analysis of the SAXS and CD data with molecular modeling and dynamics to take full advantage of the synergistic nature of the methods. This longer-term goal will involve working together with the molecular modeling and high-performance computing communities and create opportunities for new science throughout the lifetime of the beamline.

Schematic overview of the Macromolecular Characterisation beamline, the figure shows radiation emitted from a bending magnet on the left. UV is deflected upwards from the direct beam position and UV and white beam traverse the storage ring wall entering two separate monochromators. UV light and monochromatic x-rays converge on the sample position via some optical components. Appropriate detectors and a sample delivering HPLC are shown on the left.

Download Full Draft Project Proposal Here (pdf, 877kb)

References
[1] B Wallace & R W Janes, Curr Opin Chem Biol 5 (2001) 567-571.
[2] Daresbury SRCD Beamline Homepage

Contact Professor Mibel Aguilar
Position: Department of Biochemistry & Molecular Biology Monash University
Phone: +61 (0)3 9905 3723
Fax: +61 (0)3 9905 9500
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Homepage:  http://www.med.monash.edu.au/biochem/staff/aguilar.html

Contact Matthew Wilce
Position: Monash University
Phone: +61 (0)3 9902 9244
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Further Information

• Contact Ian Gentle or Garry Foran at This e-mail address is being protected from spambots. You need JavaScript enabled to view it.