Cryo-electron microscope for structural and cell biology

Unlike conventional electron microscopy (EM), cryo-EM samples are neither dehydrated nor stained and so the structure of samples remain close to the shape for the hydrated structure in the native environment. Large macromolecules and subcellular structures are proving to be excellent targets for cryo-EM. We are applying for funds for the acquisition of a cryo-electron microscope (cryo-EM) to transform the impact of our research in structural molecular biology and cell biology. Recent advances, which include direct electron detectors and data processing to correct for molecular movement in the electron beam, have revolutionised cryo-electron microscopy making it possible to determine atomic resolution structures of macromolecules and nano-machines. The results will be transformative for research in structural biology and cell biology at Queen Mary University of London. This proposal is especially timely as we have recently employed structural biologists who need access to cryo-EM (Darbari, Garnett, Milanesi), just secured access to a Titan Krios for high-resolution data collection (Pickersgill co-I), and we have a number of active and of developing projects in healthy ageing through the lifecourse, combating antimicrobial resistance, synthetic biology and bioenergy for which cryo-EM will provide a step-change in understanding the biology of the systems. We also have several existing staff who need to access cryo-EM, both single particle analysis and cryo-tomography to allow ultrastructural imaging. Finally, we have four further appointments to make in these areas in 2017/18. The equipment will also transform our ability to train the next generation of researchers, both our undergraduate students (MSci) and PhD students including those from the BBSRC LIDO DTP.

Advances in detector technology and image analysis algorithms have made cryo-EM the structural method of choice for large macromolecular complexes especially those with intrinsic dynamic properties and those available in only small quantities. With the structural biologists at QMUL investigating larger complexes and assemblies, we urgently need an instrument to extend our work using negatively stained samples to higher resolution and the combination of this medium-resolution cryo-EM plus the recently gained funding for a consortium Triton Krios will provide these much needed state-of-the-art facilities.
X-ray crystallography is a wonderful method, but with cryo-EM we can use 100-1000 times lower protein concentration and we do not have to lock our complexes into a single conformation. Since each particle is imaged individually, and can be sorted in silico according to their conformation, even highly dynamic proteins and complexes can now be captured and processed independently using cryo-EM.
Similarly our cell biologists make great use of fluorescence microscopes, their research can be extended in resolution using cryo-tomography. This facility will enable thin slices, or ultra-thin slices produced at the national facility, to be imaged using the medium-resolution microscope. When suitable samples are produced these can then return to the national facility for higher-resolution cryo-tomography studies as needed to answer the research question. For correlative light-microscopy electron-microscopy (LM-EM), a DeltavisionTM deconvolution microscope for live-cell imaging and cryotome for serial sectioning are available in the School.

CryoEM was celebrated as Method of the Year in 2015 by Nature. CryoEM advances are benefiting cellular ultrastructural imaging using cryoET (Methods to Watch Commentary, Nature Methods, Dec 2016). Thus, in situ structural biology on a proteomic scale is expected to become a reality and by investing on a cryoEM, BBSRC would enable QMUL researchers to be part of this new revolution in biological research.

Structural biology and cell biology researchers at Queen Mary University of London are major beneficiaries, this includes PIs, PDRAs, PhD students and MSci students who will all gain access to local cryo-EM facilities and by use of these facilities, state-of-the-art national and LonCEM consortium facilities.

Our collaborators, both academic and industrial, will also benefit from the more transformative science that we will be able to deliver because we will be able to study complexes produced in low quantities and dynamic and heterogeneous complexes not accessible to our current structural techniques of X-ray crystallography and NMR spectroscopy.

Pioneering work in cell and molecular biology across the University relating to the healthy organism (School of Medicine and Dentistry in addition to School of Biological and Chemical Sciences) will translate to structural studies by cryo-tomography and single particle work conferring greater impact to our outputs.

Our industrial collaborators will benefit from greater insights into the organisation and architecture of large biological systems providing greater opportunities for translatable discoveries in the biosciences. These currently include: Unilever, Sanofi, Isogenica, Prozomix, British Petroleum and GSK who support studentships.

Once a year the management team will organise a cryo-EM research day to promote the facility, the results that are flowing from it, and results from use of the national and LonCEM facilities that the cryo-EM facility allows. There will be work presentation slots for students and postdocs in addition to PIs and an external speaker from LonCEM or beyond. Through Qreview we record and publicise these talks to a wider audience (?) This meeting will serve as a platform for showcasing our enthusiasm and results in structural biology and in cell biology and will be open to academics, industry and the public.