Instruct-UK Annual Report Highlights and New Technologies
Instruct Centre UK hosts a range of technologies across seven sites throughout the UK, including Astbury Biostructure Laboratory University of Leeds (cryo-EM, NMR and hydrogen deuterium exchange mass spectrometry), Diamond Light Source (cryo-EM, X-ray diffraction, Bio-SAXS and XChem fragment screening), Research Complex Harwell (Membrane protein production and crystallisation), and facilities located within the University of Oxford in STRUBI (including the Oxford Particle Imaging Centre, OPIC) and the Department of Chemistry, which hosts the Oxford Mass Spectrometry Centre.
Find out more about the Instruct-UK Centre and apply for access to their technologies here.
Technological Advances in 2022
Harwell Campus
Electron Bioimaging Centre (eBIC)
During 2022, eBIC completed the upgrade to Aquilos 2 with autoTEM to allow semiautomated lamellae milling with overnight hold time under cryo conditions. Additionally, a vacuum Meteor fluorescent microscope was also installed on Aquilos to allow accurate targeting during lamellae preparation. Krios 3 was upgraded to include a Selectris-X falcon 4i upgrade giving state of the art energy filtered cryoET data collection, while data collection software Tomo 5 was updated to include features that improve cryoET data collection workflow and data collection speed like search maps (equivalent to medium magnification montaging in serial EM) and image/beamshift collection (potential 5-10-fold throughput increase). Automated pipelines have been developed that upon transfer with Murfey enable motion correction, restacks, alignments and 3D reconstruction and denoises tomograms.
Membrane Protein Laboratory
The new round of funding support for The Membrane Protein Laboratory (MPL) from Wellcome began in May 2022. The focus of this next phase is to augment access routes for integrative approaches to membrane protein structural biology. Developments in the preparation of small membrane protein (100kDa) samples for cryo-EM have been applied to the MPL pipeline in collaboration with eBIC. Funds were secured to purchase the Fida1 (FidaBio) which using flow-induced dispersion analysis enables protein: protein interactions and sample quality to be assessed in solution prior to preparing crystallisation plates or the preparation of samples for small-angle X-ray scattering (B21) or single-particle cryo-election microscopy (eBIC). The Fida1 will be available to the user community through Instruct-ERIC in the near future.
Diamond Light Source
New undulator sources for beamline I04 and I04-1 have increased photon flux to in excess of 10e12 photons/s and increased the scope of samples that can be studied. A significant advance has been made in data analysis for multi-crystal data collections through the implementation of xia2.multiplex at Diamond. Xia2.multiplex integrated into our automated pipelines and is have a major impact in the analysis of multi-crystal data sets from, for example, small wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipid mesophases. Unattended automated data collection now provides rapid high-quality results and is increasingly popular with academic and industrial users alike with >180,000 samples investigated since its implementation in 2020.
University of Oxford
Oxford Mass Spectrometry Centre
We moved to the Kavli Institute for Nanoscience Discovery in 2021 and now have a very high performance 1500 sq ft mass spectrometry lab, equipped with a set of instruments dedicated to the analysis of proteins and their assemblies, as well as other instruments set up for proteomics, lipidomics and glycomics. The laboratory is temperature regulated and provides a safe work environment. Two lab managers keep the facility running smoothly and take care that the instruments are running smoothly. Highly skilled scientists perform high-throughput data analysis and support visitors with the measurements. We also have access to a state-of-the-art wet lab with additional facilities covering all aspects of molecular biology, providing sample preparation space. Our cold room and several -80 freezers have capacity to receive samples for remote use of the facility.
Oxford Particle Imaging Centre (OPIC)
In 2022, OPIC acquired the PRIMO (alveole) Micropatterning device for improving the positioning of cells on grids. This has greatly improved the number of 'usable' cells for FIB-milling and regular cryo-ET in the cellular tomography workflow. OPIC also now has an iFLM attachment on ourtheAquilos2+ FIB-SEM for correlative experiments, and now have the capacity to mill samples overnight, thereby greatly improving throughput.
UK Structural Biology Activities 2022
OPIC hosted an intern from the Equipe Moali group (Université de Lyon, France) who specialise in metalloproteases and Tissue Remodelling in the Tissue Biology and Therapeutic Engineering. Arriving with purified samples, she learned cryo-EM from scratch. During her stay, the intern optimised sample and grid preparations and learned to collect and solve cryoEM structures. By the end of her visit, she achieved a final reconstruction around 4 A resolution, which was a great success given that it was a small highly flexible protein complex, mostly comprised of beta-sheets and a manuscript is now in preparation. Additionally, follow-up structure-based experiments have been planned.
Other UK Structural Biology events and initiatives:
- Frontiers in Native Mass Spectrometry and Single-Molecule Imaging (MS+M_22) Oxford August 2022
- Serial Crystallography Workshop 2022 at Diamond Light Source
- SAXS Fundamentals Training 2022
- Diamond-CCP4 Data Collection and Structure Solution Workshop 2022
- Early Career Scientists Symposium 2022
- CCP-EM Spring Symposium 3-5th May 2022
- CCP4 Study Weekend (Virtual) 5-7th January 2022
- Biochemical society 3rd Annual workshop on Membrane Proteins
- The Astbury Conversation 2022 (virtual)
