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High Performance Macromolecular Crystallography Beamline (MX3)
BRIGHT Beamlines

High Performance Macromolecular Crystallography Beamline (MX3)

The High Performance Macromolecular Crystallography beamline (MX3) will be capable of providing high-flux, microfocus X-rays for small and weakly diffracting protein crystals. The beamline will provide three modes of operation: goniometer, serial crystallography and in-tray screening. The beamline will be powered by a 3m in-vacuum undulator. The proposed optical system will maximise flux at the sample position and the use of a secondary source aperture will allow rapid change in beam size. The goniometer will be able to be translated out of focus to allow increase of beam size without changing the optical components. In order to produce the required high flux the beamline will also utilise a double multilayer monochromator. This will increase the bandpass (energy width) and brightness of the X-ray beam; however, this will mean that Multi-wavelength Anomalous Dispersion (MAD) experiments will not be feasible.  The beamline will specialise in high flux, high speed data collection on micro crystals with a high degree of automation for crystal location and data collection.

Samples

The MX3 beamline will accept macromolecular crystal samples in several formats.

Sample shipping for robot-compatible pins will be identical to other MX beamlines at the synchrotron and remote access will be available. In-tray screening may also be provided via a mail-in system.

The core measurement capabilities of MX3 include:

  • Single-pin goniometer experiments:
    • “Standard” MX collection on robot mounted single crystals. Due to the high flux of MX3, an attenuated beam will be required to collect a full dataset from a single crystal, or multiple partial datasets merged from several crystals.
  • In-tray screening:
    • Automated in-tray screening using a tray holder on the goniometer.
  • Fixed-target:
    • High throughput fixed target collection using silicon chip-based sample holder. Chips will be loaded offline, aligned on the beamline (separate fixed-target stages) and collected.
  • Injector collection:
    • Crystals introduced to the beam via a sample injector.  Several designs are being considered but the production system will deliver crystals to the beam with high speed and low background scattering to allow data collection and structure solution from multiple micro-crystals.

Scientific Applications

MX3 will allow experiments that are currently only marginally feasible or impossible on the existing MX1 or MX2 beamlines (such as small GPCR crystals or the use of fast injectors/serial crystallography). Such a beamline will enable Australian researchers to keep pace with the advances in the field of synchrotron-based crystallography. MX3 will address the following current and predicted needs of the crystallographic community in Australia, New Zealand and Asia:

  • The unmet need for high-flux microfocus crystallography.
  • The unmet need for serial crystallography (both at cryogenic and room temperature) this will allow researchers to overcome a current bottleneck in the production of crystals suitable for data collection (currently being implemented on MX beamlines worldwide). As well as the merging of multiple datasets from these samples into a ‘complete’ dataset. I.e. poorly diffracting samples, large protein complexes, etc…
  • The unmet need for an ultra-high throughput facility for drug design and fragment screening.
  • In-tray screening. The rate-limiting step in macromolecular crystallography is the production of crystals suitable for diffraction experiments. In-tray screening is a technique for rapidly assessing crystallisation trays to find conditions and micro-crystals using X-ray diffraction. This can greatly reduce the time required to produce crystals that are suitable for data collection.

Operation of MX3 will allow MX1 to specialize in low-energy biological macromolecular crystallography (MX) and chemical crystallography (CX) collection. The current MX1 and MX2 beamlines cater for the needs of CX users providing relatively high flux small beams (few CX samples are flux limited).  Future new capabilities for synchrotron-based chemical crystallography will be focused on in-situ environments such as furnaces and pressure cells. CX users will also have access single crystal diffraction capabilities using the Advanced Diffraction and Scattering (ADS) beamline where higher X-ray energies will allow for effective use of large experimental systems such as furnaces and pressure cells.

MX3 will provide the ability to collect single crystal diffraction data at very high speed; this is needed in several instances:

  • Membrane proteins and protein complexes: As these types of projects often result in small visually invisible and weakly diffracting crystals, it is often necessary to screen hundreds or thousands of crystals in order to find a crystal suitable for data collection (or to merge data from many crystals).
  • Rational Drug Design (Fragment Screening): In this example a library of compounds (from many hundreds to thousands) are soaked (or co-crystallised) into crystals of the protein of interest. The proteins used in fragment screening studies must already be shown to crystallize readily with suitable diffraction qualities (resolution/survivability in the beam). With a library containing 500 fragments it would be necessary to collect 1000+ datasets (to ensure each fragment is visualized).
  • Room Temperature Diffraction: The diffraction quality of each and every crystal hit can be automatically evaluated, thus removing human error. As the cryo-cooling of crystals can change or destroy them, any diffraction observed from room-temperature diffraction experiments is a true representation of the crystal’s nature. Software has already been implemented on the existing MX1 and MX2 beamlines that evaluate the quality of the data and rank the quality of the crystals based on the diffraction images. 
Membrane protein/receptors

The MX3 beamline will be particularly useful in the study of proteins that are difficult to purify or have complex or changeable structures; for example, proteins that are located in or associated with membranes. Normally, such proteins present enormous challenges with respect to crystallisation and therefore structure determination. This is particularly true for receptors, which are present within membranes. Protein receptors are a crucial part of all cell signalling pathways, where dysfunction can lead to a range of diseases including many forms of cancer. Most membrane receptors exist as complexes of smaller proteins, called subunits, which can only be purified in very small quantities as microcrystals. These crystals cannot be studied on the current MX beamlines available at the AS but could be analysed using MX3.

A specific disease case study relates to Alzheimer’s disease. This neurodegenerative disorder is characterised by the presence of extracellular amyloid plaques in the brain. In the quest for a cure for this condition, Australian scientists are studying precursor proteins that are responsible for the peptides that form the plaques and have been shown to be toxic to neurons. Only very small crystals of these targets have been produced, which are too small to study with the current AS beamlines.

Another example is the study of biological rotary motors. These are fascinating protein assemblies that are found in the body and which are inherently difficult to study due to their trans-membrane nature and size. Once again, facilitation of this study is critical as these structures are often implicated as the cause of diseases, such as heart disease. A high-brilliance microfocus beamline, such as MX3, will allow scientists to expose only small parts of a crystal that might be better ordered than others. Currently Australian researchers from the Victor Chang Cardiac Research Institute have been forced to work at the Advanced Photon Source in the USA, and the European Synchrotron Radiation Facility in France in order to undertake such studies.

Virology

Viruses are responsible for a wide range of diseases in humans and other animals, ranging from the COVID-19, common cold to ebola, AIDS and even some types of cancer.  Aspects of viral structure and behaviour have been studied using crystallography, but this normally involves small, weakly diffracting crystals which will be able to be studied using MX3. For example, insect viruses can remain active for years in the environment using a type of armour formed of crystals called viral occlusion bodies. Understanding these bodies would have important implications for the development of new bio-insecticides and vaccines. A wide range of other applications exists in the life sciences, and this need is increasing as scientists turn to the more challenging but potentially more significant health problems that this beamline will make possible.

Materials science

A wide range of materials only form incredibly small crystals that cannot be studied using existing beamlines. Examples include microporous and mesoporous materials, hydrogen storage materials, novel metal oxides and ceramics, superconductors, minerals, 'smart' materials, piezoelectric materials, novel magnetic materials, photonic devices, information storage materials, molecular switches and sensors, biomimetic materials and pharmaceutical materials.

Technical Information and Specifications

The MX3 beamline is designed to provide the highest flux in a microfocus MX beamline available at the Australian Synchrotron. This is achieved through the use of an in-vacuum undulator (IVU) along with the use of a double multilayer monochromator (DMM) and horizontal secondary source aperture (SSA) provide the high flux and small focal size, respectively.

Beam stability is a key design requirement with the only vertical optical element being the vertical focusing mirror (VFM) in concert with the horizontal focusing mirror (HFM) adjacent to the sample. This should reduce the impact of vibration on beam position given the use of a horizontal DMM and horizontal SSA.

Thermal stability in the endstation is critical with a design goal of 0.1°C rms in the endstation and optics. Humidity will be controlled and as low as 25% for routine robot operations.

A large pixel-array detector (such as an Eiger2 16M) will be used.

Options for goniometers and sample loading robots are being evaluated.

Beamline Layout

MX3 beamline layout

Technical Specifications

View MX3 Technical Specifications

Energy range

10 to 14.5 keV

Number of endstations

1

Source

Type

3m In-vacuum undulator (IVU)

 

Pole pairs

169

 

Period

1.75 cm

 

Total power

2.34 kW

Optics

 

 

 

Horizontal double multilayer monochromator

Ru/B4C with 3 sets, 0.9% bandpass, 0.5% and potentially 0.3%.

 

Horizontal prefocussing mirror (PFM)

600mm long at 3mrad

 

Horizontal Secondary source

170 micron gap (FWHM) for full beam. Down to 34 micron gap for 2x2 micron beam.

 

Vertical focussing mirror

400mm long at 3mrad

 

Horizontal focussing mirror

400mm long at 3mrad

 

 

 

Spot size at the sample position

Full beam 8-2 microns (FWHM) at > 6e13 ph/s

 

Minimum beam 2x2 microns (FWHM) at > 1e13 ph/s

 

Beamline status

View MX3 beamline status

Scheduled Completion Dates

Key Project Deliverables

July-2019

Project Started

2020

Design Completion

2022

Procurement Completion

2022

Equipment Installation Completion

2022-2023

Cold Commissioning Completion to 1st Light

2023

Hot Commissioning Completion, includes expert users

*Q3 2023

First User Experiments; Beamline fully commissioned over the next 12 months

*Delay due to covid-19 considered unlikely

Staff

Dr Nicole Cain – Project Manager

Dr Tom-Caradoc-Davies – Lead Scientist

Mr Hima Cherukuvada – Lead Engineer

Beamline Advisory Panel

Professor Charles Bond (Chair) – University of Western Australia

Associate Professor Peter Czabotar – Walter and Eliza Hall Institute of Medical Research

Dr Gwyndaf Evans – Diamond Light Source, UK

Dr Aina Cohen - SLAC National Accelerator Laboratory, USA

Professor Emily Parker, Victoria University of Wellington, NZ

Professor Chris Sumby, University of Adelaide

Associate Professor Stephanie Gras, Monash University

Contact

as-mx3@ansto.gov.au