Core Technology Facilities

Mass spectrometry based proteomic experiments have become an important tool for studying biological systems. The goal of the Biological Mass Spectrometry and Proteomics Laboratory is to implement state of the art mass spectrometric techniques to provide scientists at the LMB with the capabilities to identify, characterise and quantify proteins present in a particular network, pathway, organelle, cell or tissue.

The Biological Mass Spectrometry and Proteomics Laboratory principally employs electrospray ionization mass spectrometry as the key technology for both qualitative and quantitative characterisation of proteins. However, the Laboratory has access to several mass spectrometric technologies, including MALDI, thereby allowing investigators to adopt multiple analytical strategies. The basic approach for protein analysis is ‘bottom-up’ proteomics, where proteins are initially enzymatically digested with proteases producing a peptide pool representative of the original proteins.

J Mark Skehel is Head of the Biological Mass Spectrometry and Proteomics Group.

The LMB has world class facilities for biophysical techniques, run by an expert team with many years of experience over a broad range of techniques and in many different applications.

Biophysics is an interdisciplinary science that applies theories and methods from the physical sciences to questions in biology. Biophysical techniques can contribute to all areas of structural biology from expression and purification issues through to high resolution structure and can probe interactions and biology from single molecules through to whole cells. These methods have been central to the success of many research programmes in the LMB. Researchers can access the most common biophysical techniques (AUC, ITC, DSC, CD, LD, MALS, QELS, SPR and fluorescence) and a number of ‘in-house’ instruments for performing the latest cutting edge techniques in rapid reaction kinetics or single molecule spectroscopy.

To help researchers to make their own routine measurements, the biophysical facilities team provides training and runs an annual lecture series on methods and instrumentation within the LMB. In addition, team members are available to discuss proposed experiments and advise on the results of routine experiments; and they can also form collaborative projects, where there is a requirement for specialist skills in the design and execution of the work and/or in complex data analysis.

The biophysical facility is run by Chris Johnson and Stephen McLaughlin.

The LMB has an outstanding electron microscopy (EM) facility, the focus being on electron cryomicroscopy of unstained biological material. The LMB has five EMs available, of which four are equipped for work with frozen specimens and two have 300kV FEG electron sources. The facility’s most recent acquisition was an FEI Polara microscope with energy filter, which has greatly facilitated electron cryo-tomography. Equipment for high pressure freezing and cryomicrotomy is also available, allowing cryoelectron-tomographic analysis of vitreous sections.

The facility is primarily intended for hands-on research by students, postdocs and group leaders, who prepare specimens, operate the EMs and analyse the resulting images or diffraction patterns themselves.

Future plans include implementing technical developments that can improve the quality of cryoEM structures and replacing older microscopes to tie in with the move to the new building.

In addition to the in-house equipment, members of the LMB are part of the international electron microscopy community and have access to additional equipment in other laboratories around the world, on a reciprocal basis.

The electron microscopy facility is managed by Shaoxia Chen.

The LMB’s flow cytometry facility gives its researchers the ability to characterise, quantify and separate cell populations on the basis of surface and intracellular markers.

The facility provides researchers with both a service and training in new techniques. For example, it provides advanced flow cytometry applications, such as DNA cell cycle analysis, cell proliferation and apoptosis measurements, calcium flux measurements, and multi-colour immunofluorescence analysis. It also supports innovative techniques such as sorting of particles, emulsions or bacteria and provides a routine cell sorting service including single cell cloning, rare event sorting and high speed sorting.

Users of the facility have access to eight flow cytometers. There are three high-speed sorters capable of sorting at rates of up to 70,000 cells per second (two Sony Synergy systems and one Beckman Coulter MoFlo). There are five analysers (one Fortessa, one LSRII, one Eclipse, and two FACSCaliburs).

Maria Daly, assisted by Fan Zhang, heads the flow cytometry facility. The team works under the scientific oversight of Cristina Rada, a joint programme leader in the PNAC Division.

Digital image processing is an essential step in the determination of macromolecular structures by electron microscopy. Over the years LMB staff have developed a special system of image processing programs to determine macromolecular structures, which is now used, in various forms, around the world.

The centrally important procedures are the averaging of many images of the sub-unit to improve the signal, the correction for various transfer functions, and the generation of a three-dimensional map from a set of two-dimensional projections. The detailed way in which these computational procedures are best carried out depends on the symmetry of the object and the type of specimen preparation.

The LMB now has a set of (over 80) programs, written by various members of the LMB, for processing images of two-dimensional crystals and of particles with helical or icosahedral symmetry. The LMB’s philosophy has been to write stand-alone programs and to give the whole system coherence by adopting standard formats for the storage and interchange of different kinds of data.

The Image processing system is managed by Jude Short.

The modern form of the confocal microscope was developed at the LMB in the mid-80s, and instrument development continues today. Light microscopy is central to the work of many groups at the LMB and the move to a new building has allowed the Laboratory to develop a more centrally organised light microscopy facility with additional support for microscopes belonging to individual groups.

The new LMB building provides 18 microscopy rooms distributed throughout the divisions, as well as a centralised light microscopy facility housing advanced and specialised microscopes. This facility houses three new super resolution microscopes (using structured Illumination, single molecule localization and stimulated emission depletion) offering image resolution below 50nm in suitably prepared samples. In addition, LMB researchers can access specialist, advanced equipment in other local laboratories.

The Light Microscopy Facility is headed by Nick Barry, an optical physicist and specialist in light microscopy.

The newly-established microfluidics facility has a Neutronix Quintel 4006 mask aligner, spin-coater, plasma oven and all the equipment necessary for the fabrication of PDMS chips with feature sizes down to a few microns. The facility is available to users after a training session.

Microfluidics fabrication is managed by Paul Dear.

The facility’s operational model is to deliver a biology-driven facility that complements other structural and biophysical techniques. The facility offers both high-end research collaborations to answer specific biological questions and maintains the automated routine service NMR for synthetic chemistry.

Relevant advanced solution state NMR methods are implemented and can be adapted to the specific needs of LMB projects. Supported key research areas include ubiquitination and autophagy, function and dynamics of multidomain complexes and functional studies of membrane proteins.

The state of the art NMR facility – located in a separate, purpose-built building – houses 500, 600, 700 and 800 MHz spectrometers, all equipped with cryoprobes and multichannel configuration. It also provides robotics for screening small molecule compound libraries against potential drug targets.

Stefan Freund is Head of the NMR Facility.

Scientific Computing provides and supports the LMB’s core Unix systems. These range from computer servers providing email, to authentication and controlled access to the LMB network, to remotely accessible Unix machines, desktop workstations, and a 700 CPU High Performance Computing (HPC) cluster.

Staff from the Scientific Computing team are available to provide advice on computational problems and access to a wide range of scientific software is freely available.

Scientific Computing is headed by Jake Grimmett.

The LMB’s transgenic mouse facility produces transgenic animals by pronuclear microinjection. It can also carry out injection of targeted embryonic stem cell clones into mouse blastocysts, after which the chimeras are bred at the Ares facility to establish germline transmission of the allele.

The facility also provides a high-throughput genotyping service for the LMB.

The transgenic mouse facility team is led by Lesley Drynan under the scientific oversight of Andrew McKenzie.