Membrane Protein Laboratory (MPL)
The Membrane Protein Laboratory (MPL) at Diamond Light Source is a research and training user facility open to scientists from laboratories anywhere in the world that are interested in Structural Biology of Membrane Proteins. The laboratory combines recently developed high throughput technologies for protein production and crystallisation with the latest developments in X-ray diffraction data collection systems at Diamond's Macromolecular Crystallography (MX) beamlines.
Membrane proteins are important pharmaceutical targets with 60% of the current marketed drugs targeting this class of proteins. However, a major challenge in designing drugs to target membrane proteins is the need for high resolution structural information. With the MPL situated just metres from Diamond’s Microfocus MX beamline (I24), world-class expertise and tools are brought together to tackle this challenge.
HTP Protein Production
The purification platform facility is composed of 150m2 of wet laboratory space at the Research Complex at Harwell (RCaH). The RCaH wet laboratory is operational with cutting-edge technology and equipment for the expression and purification of membrane proteins.
Yeast cells with GFP and His fusion tag construct are available for membrane protein expression at the MPL. Protein purification typically includes immobilised metal affinity chromatography (IMAC) using His-tag and size exclusion chromatography (SEC). The most suitable detergent(s) for the protein extraction are selected based on results from fluorescence-detection size exclusion chromatography (FSEC) using GFP tag. Subsequently, the protein(s) are characterised by size exclusion chromatography with multi-angle light scattering (SEC-MALS) before crystallization trials.
The equipment available at the MPL HTP purification platform includes:
- Six refrigerated orbital shakers;
- One cell disrupter with a cooling system;
- Three floor standing ultracentrifuges;
- Two floor standing centrifuges;
- One benchtop ultracentrifuge;
- Two benchtop centrifuges;
- Four AKTA purifiers with associated columns;
- One fluorescence spectrophotometer/plate reader;
- One Nanodrop spectrophotometer (ND-100);
- One Fluorescence-detector size exclusion chromatographic system and other small equipment.
The crystallisation facility is composed of 140 m2 of wet laboratory space at Diamond in the synchrotron building. The facility is adjacent to the microfocus beamline I24.
Methods available for crystallisation of membrane protein(s) are crystallisation from lipidic cubic phase (LCP) and vapour diffusion with sitting drops. The MPL uses commercially available crystallisation screens (MEM GOLD, MEM GOLD2, MEM MESO, MEM START and MEM SYS) as well as MPL customised screens for initial crystallisation screening.
The equipment available at the MPL HTP crystallisation platform includes:
- One SEC-MALLS system (Malvern);
- One TTP Mosquito LCP;
- One Topaz crystallisation system;
- Two automatic imaging systems (4C and 20C) with barcode printers;
- One LCP-FRAP system (Fluorescence Recovery After Photobleaching);
- Three microscopes + 2 digital cameras;
- One Hamilton Star lab Liquid Handler;
- Two automatic sealers;
- Three crystallisation incubators;
- Several storage dewars and other small equipment.
Data Collection Systems
Membrane protein crystals, especially those produced via the Lipidic Cubic Phase (LCP) method, are often very small, extremely fragile, poorly ordered (high mosaicity) and very sensitive to radiation damage. Collecting data from such crystals is not a straightforward task and the resolution of the X-ray diffraction data not only depends on the crystal quality but also on the beamline specifications.
The proximity of the X-ray source to the MPL provides a unique advantage in alleviating problems in crystal transport and facilitates a quick turnaround between design of an experiment and its execution. Diamond provides a platform combining recently developed high throughput technologies for protein production and crystallisation with the latest developments in X-ray diffraction data collection systems. This allows our users to characterise early stage crystal hits and swiftly determine their promise for optimisation trials leading to a rapid project progression.
Facilities available at Diamond include:
- State-of-the art membrane protein expression and crystallisation platform;
- Support by highly experienced scientists in the field;
- Microfocus MX beamlines (I24 and I04);
- Fast grid scan on I24;
- Powerful processing pipelines (Blend, Dials, Xia2…);
- In situ crystal screening using the I24 microfocus beamline;
- In situ crystal dehydration using the I24 microfocus beamline.
The Industrial Liaison Team at Diamond
We’re always happy to discuss any enquiries or talk about ways in which access to Diamond’s facilities may be beneficial to your business so please do give us a call on 01235 778797 or send us an e-mail. You can keep in touch with the latest development by following us on Twitter @DiamondILO or LinkedIn