Services & platforms available

The success of membrane protein structural analysis largely depends on the quality of protein samples. We offer a large portfolio of biophysical methods which support the good quality control of protein samples produced. These options are designed to quickly help you identify the most suitable protein constructs and purification conditions, followed by protein characterisation. Proprietary clients can enter the pipeline at any point providing the protein sample passes the quality control step confirming that it is suitable for subsequent steps of the platform.

Protein samples are processed using HTP extraction and purification screens. The most suitable conditions are selected based on results from SDS-PAGE and Fluorescence-detection Size Exclusion Chromatography (FSEC).

Membrane protein purification and formulation allows scientists to screen a wider range of conditions, including but not limited to detergents, amphipols, membrane scaffold proteins (MSPs), SMALPS, lipids, pH and salts. A typical purification process utilises Immobilised Metal Affinity Chromatography (IMAC) using His-tag, a reverse IMAC after construct cleavage (TEV or 3C protease), and Size Exclusion Chromatography (SEC).

Protein quality is confirmed using SEC-MALS and a thermostability assay (providing the protein is sufficiently pure (>90%)).  If a simple assay is available, the target protein function could be tested at this stage. For example, if the target protein substrate is known, then Nano DSF could be used to check for a protein-substrate interaction. 

The Crystallisation Facility (CF) at Diamond offers services for both crystallisation and crystal harvesting for X-ray diffraction data collection. Co-located alongside the Diamond synchrotron, CF services are an integrated part of the full-service structural biology package available for industrial customers at Diamond. This includes X-ray crystallography and structural analysis of protein targets. 

High throughput robotics are available for protein crystallisation at the Diamond Crystallisation Facility. The platform allows crystallisation of both soluble protein samples as well as membrane protein crystallisation in Lipidic Cubic Phase (LCP).

Crystallisation screening is offered using common commercial course screens as well as custom screens which can be prepared using the Formulator liquid handler. Mosquito (SPT Labtech) and Gryphon (Art Robbins) robotics are available for the preparation of sitting drop and LCP crystallisation plates. The Formulatrix Rock imager 1000 imaging systems offers a range of crystallisation temperatures and have cross-polarisers, UV and multi-fluorescence imaging options. These images can be accessed remotely. 

Purified and characterised proteins can be used for initial crystallisation screening using commercially available screens. Vapour diffusion crystallisation is available at the following temperatures: 4^oC, 12^oC, 16^oC, 19^oC and 20^oC.  

Examples of the commercial screens offered at the platform include:  

• _{BCS Screen (Molecular Dimensions)}  
• _{SG1^TM Screen (Molecular Dimensions)}  
• _{Morpheus^® I-III Screens (Molecular Dimensions)} 
• _{JCSG Plus Screen (Molecular Dimensions)}  
• ^{ProPlexTM Screen (Molecular Dimensions)}  
• ^{PGA ScreenTM screen (Molecular Dimensions)}  
• ^{Structure Screen 1 & 2 (Molecular Dimensions)}  
• ^{Crystal Screen 1 and 2 (Hampton Research)}  
• ^{WizardTM Classic Screen (Molecular Dimensions)}  
• ^{Wizard CryoTM Screens 1 & 2 (Molecular Dimensions)} 
• ^{Clear Strategy ScreenTM I CSS-I (Molecular Dimensions)} 
• ^{PACT PremierTM Screen (Molecular Dimensions)} 
• ^{Grid Screen MPD (Hampton Research)}  
• ^{PEG/Ion Screen (Hampton Research)} 
• ^{NeXtal Classic (Qiagen)}  
• ^{Ligand-Friendly Screen - LFS (Molecular Dimensions)}  
• ^{LMB Crystallisation Screen (Molecular Dimensions)} 

Prior crystallisation of membrane proteins in the LCP Fluorescence Recovery After Photo Bleaching in LCP (LCP-FRAP) instrument allows pre-screening for likely conditions that may lead to membrane protein crystals.

The fluorescently labeled proteins (cy3 dye) are irreversibly photobleached using laser power. The subsequent diffusion of the surrounding non-bleached fluorescent molecules into the photobleached region is then monitored. If the protein does not diffuse it will also never crystallise. Therefore, LCP-FRAP is a great tool to accelerate the membrane protein crystallisation process, by eliminating unsuitable conditions before the lengthy process of crystallisation screening and optimisation is attempted.

 Read our LCP-FRAP case study to find out more

Two crystallisation methods - LCP crystallisation and vapour diffusion crystallisation with sitting drops – are available. The initial crystallisation screening is offered using commercially available screens:

• ^{JBScreen LCP (Jena Bioscience)}
• ^{MemGoldTM, MemGold2TM}
• ^{MemMesoTM, MemStartTM and MemSysTM (Molecular Dimensions)}
• ^{MPL-customised screen for LCP}

For ion channels and transporters, the following are also available:

• _MemChannel^TM
• _MemTrans^TM _{screens (Molecular Dimensions)}  

Crystallisation conditions from initial crystallisation screens can be optimised to obtain single crystals with dimensions and quality suitable for X-ray diffraction tests on Diamond microfocus beamlines (cryocooled or in situ). If any X-ray diffraction has been observed during the X-ray diffraction screening step, these results can be used to further guide the optimisation of the crystallisation conditions. 

Crystal harvesting and cryoprotection of the harvested crystals in a suitable cryoprotectant are available for single crystals of a size and quality suitable for X-ray diffraction data collection that have been obtained from the crystallisation trials. The harvested crystals in the Unipucks are available for beamtime scheduled at Diamond macromolecular crystallography (MX) beamlines.

Data collection of X-ray diffraction images from harvested protein crystals is a part of the structural biology package offered to Diamond’s proprietary clients. Dedicated Diamond scientists will attempt to collect the X-ray diffraction data set if diffracting protein crystals are identified. The data wedges obtained from individual crystals will be merged until a complete data set is obtained. 

Services and access modes available for MX at Diamond

If a good homology model structure is available, an attempt to solve the protein structure by molecular replacement (MR) methods will be made by Diamond scientists. Alternatively, experimental S-SAD phasing is available for projects with no suitable model structure. Successful structural analysis will be completed by structural model refinement.