bio-systems by industry Jitka Waterman Diamond Light Source EUCALL - PowerPoint PPT Presentation

Use of large scale facilities for study of bio-systems by industry Jitka Waterman Diamond Light Source EUCALL Workshop: Biology at Advanced Laser Light Sources 30 th November – 1 st December 2017

Harwell Science & Innovation Campus MRC ISIS - neutrons PHE Central Research Laser Facility Complex Satellite Applications Catapult RAL Space Diamond Light Source E6 ESA

Diamond Light Source Overview • Largest scientific facility built in the UK for over 40 years • Diamond is a non-profit private company formed as a joint venture between STFC (86%) and The Wellcome Trust (14%) • All beamlines are owned and operated by Diamond Phase 1 7 beamlines completed 2007 Phase 2 +15 beamlines completed 2012 Phase 3 Key Facts +11 beamlines Employee: > 500 to be available External scientists: > 3000/year by 2018 Publications: ~1000 in 2015 PDB: >4000 model deposited

Industrial Liaison Office Alex Dias Renjie Zhang Sally Irvine Anna Kroner Claire Pizzey Jason van Rooyen Jitka Waterman Sin-Yuen Chang Leigh Connor Elizabeth Shotton MX XChem Imaging XAS SAXS Cryo-EM MX XAS XRPD, Engineering Group Leader • Head of ILO: Dr Elizabeth Shotton • Comprising expert scientists in a wide range of techniques • Services include:

Biopharmaceutical Industry Early Stage Drug Discovery Process Diamond – Opportunities related to structural biology Macromolecular crystallography beamlines • Platforms for protein expression and crystallisation • Fragment screening platform (Xchem) • BioSAXS and circular dichroism • Electron Microscopy (eBIC) • Imaging •

Beamlines & other facilities at Diamond I14 Macromolecular Crystallography Materials Soft Condensed Matter Engineering and Environment Spectroscopy Surfaces and Interfaces ePSIC eBIC

• 5 state-of-the-art undulator beamlines • 3 high brilliance MAD beamlines (I03, I04, I24), • a fixed wavelength beamline (I04-1) & fragment screening platform • a long wavelength (1.5 – 4 Å) beamline (I23) • microfocus beamlines (I04, I24).

Standard Experiments on MX Beamlines • RacerSnake grid scans - Microcrystals, samples in LCP, X-ray centring • Sample changer - Capacity 37 pucks (592 samples) - 17s changeover time • SmarGon - Faster and precise sample centring and sophisticated data collection with multi-axis goniometer functionality • HC1 dehumidifier - Samples with high mosaicity, poor spot profile, low resolution or large unit cell • Fluorescence detector - Spectra – probe sample for anomalous scatterer - Scans – define energies for SAD/MAD

Data Processing and Analysis No time to think • Sample exchange in <20s • Data collected in ~1m • Up to ~35 samples per hour Rapid feedback

ISPyB Information management system for tracking samples and datasets

Unattended Data Collection • BART robot • Loop centring routine − OAV or X-ray centring • Standard data collection • Jobs queued in GDA • Data auto-processed – Xia2, Dials, DIMPLE

Fragment Screening Platform (Xchem) “In crystallo ” screening of fragment libraries • First platform of its kind at synchrotron – Project initiated in 2014 – Operational since 2015 – ILO commitment from early developments • Screen hundreds of compounds in days • Automated data collection • Powerful software for data analysis – Rapid hit identification Hits from JMJD2-DA (SGC) System compatible with external libraries • Courtesy of Patrick Collins Diamond Light Source Platform associated to I04-1 (optimized OAV • automated centering)

Fragment Screening Platform (Xchem) Soaking 15-30 m compounds Crystal 1-2 days harvesting Data 1-2 days collection Hit 1/2 day identification

Microfocus Beam Applications • I24, I04,VMXi and VMXm • Study of membrane proteins, large macromolecular complexes or viruses • Serial crystallography (I24 and VMXi) – UK-XFEL Hub – Time resolved pump-probe experiments at slower time scale (ms) as an alternative to XFELs (fs) – LCP injector – sample preparation for XFEL experiment I sec exposure - 2Å data Crystal volume ~5000x smaller than 100 micron ’standard’ ‘ Class C GPCR metabotropic glutamate receptor 5 transmembrane domain.’ Doré, AS et al. Nature (2014) 511, 557-562

Long Wavelength MX (I23) Dedicated beamline for experimental phasing • Optimized for operation in 1.5 – 4 Å range • S-SAD for native proteins and P-SAD for RNA/DNAs • Element identification (Ca, Zn, K, Mn, Cd) • MAD exploiting M-edges for large complexes (U, Pt, Hg) P12M detector Main challenges • Absorption Fluorescence detector - Vacuum sample environment OAV - Analytical absorption correction Goniometer by X-ray tomography Tomography camera • Large diffraction angles Sample position - Curved detector assembly Novel in-vacuum vessel

New MX developments - VMX • Versatile Macromolecular Crystallography (VMX) is…. Two next generation MX beamlines that will provide: • A sub micron variable focus beam in the 0.5 – 5 micron range • A dedicated in situ data collection facility • VMX will deliver…. • Capabilities to solve the toughest crystallographic problems that are beyond the reach of existing experiments • Revolutionise the way the community collect data on many samples through use of in situ diffraction

VMXi - in situ data collection Image-based alignment Compact hutch design Crystallization optimisation and testing • In-situ data collection • Too small, fragile or cryo-incompatible samples • • Fragment screening (Xchem) • Samples in LCP (films) FORMULATRIX Serial crystallography Automatic sample handling •

Electron Microscopy facility Diamond I13 I14 and EM Facility Building Services

A National User Facility for Biological Electron Cryo-microscopy (eBIC) Funded by the Wellcome Trust, MRC and BBSRC at level of £15.6 M over 5 years The facility includes: - 4 high-end 300keV automated cryo EMs (Titan Krios FEI) - 200 keV automated feeder instrument (Talos Arctica) - Cryo focused ion beam instrument - Sample prep including vitreous sectioning - Correlative fluorescence/EM - FEI Polara @ Oxford for CAT 3 samples - Single particle microscopy - Cryo-electron tomography

• Supporting suite of biophysical techniques • BioSAXS on beamline B21 • Infrared spectroscopy on beamline B22 • Circular dichroism on beamline B23 • Cryo X-ray microscopy on beamline B24

In Solution Protein Characterisation BioSAXS (B21) Parameters obtained by SAXS analysis • BioSAXS sample changer • In line HPLC and MALLS Applications Validate structural hypothesis • – Determine of the size and shape of proteins in solution – Low resolution 3D structure (~15 Å) – Map different components of a complex • Characterise conformational changes – Ligand binding – Flexible proteins • Biologics (antibodies, vaccines…) – Investigate effect of different formulations – Protein stability studies Holbourn et al JBC (2011) 286 (25) 22243-22249

In Solution Protein Characterisation Circular Dichroism (B23) Applications • Protein stability and Kd determination • Spectroscopic technique to study a variety HTCD - 96-well plates – compare protein • of chiral materials - small molecules conformation under a range of conditions (drugs), polymers and biopolymers (nucleic acids, proteins, carbohydrates and lipids) • CD imaging of films - 2D mapping to assess the homogeneity of the sample preparation • Improved signal-to-noise ratio due to high flux • High pressure SRCD – measure the secondary structure content under high pressure Protein stabilisation upon binding of a metal ion Pump-probe SRCD for time-resolved studies • B23 experimental room Rajasekaran et al, Biophys. Res. Com 398 (2010)

Infrared Spectroscopy (B22) • Synchrotron infrared spans a larger spectral range extending into the far-IR region and can be 100-1000 times brighter than any other broadband IR source • IR microspectroscopy (FTIR) allows not only molecular identification but also IR imaging with very high spatial resolution and sensitivity Nucleic acid & Carbohydrate Protein Water Lipid The mapping of an individual cell by SR-FTIR Applications • A relevant method to screen tissue and cytology samples Determine cellular and sub-cellular chemical speciation in both cancer and normal cells • • Diagnosis of cancer at an early stage (when biopsy can be inconclusive)

Cryo Transmission X-ray Microscopy (B24) • 3D imaging technique  internal structure of vitrified cells • Resolution: ~30-50nm in up to ~10um thick samples • Data collection at ~500eV (“water window”) • Complementary to cryoEM and light microscopy Applications – Imaging nanoparticles, for example determine location of particles in tumour cells – Could be used to image metal nanoparticles >20nm, might be challenging to view smaller/lighter particles Nuclear membrane Mitochondria Nuclear pores ~ 120 nm in diameter Nucleus 1 μ m Nucleoli

Thank you Jitka.waterman@diamond.ac.uk http://www.diamond.ac.uk/industry.html EUCALL Workshop: Biology at Advanced Laser Light Sources 30 th November – 1 st December 2017