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 30th November – 1st December 2017

Diamond Light Source

ESA E6

RAL Space

ISIS - neutrons

Central Laser Facility

MRC PHE

Satellite Applications Catapult Research Complex

Harwell Science & Innovation Campus

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 +11 beamlines to be available by 2018

Key Facts Employee: > 500 External scientists: > 3000/year Publications: ~1000 in 2015 PDB: >4000 model deposited

Industrial Liaison Office

• Head of ILO: Dr Elizabeth Shotton
• Comprising expert scientists in a wide range of techniques
• Services include:

Elizabeth Shotton Group Leader Anna Kroner XAS Leigh Connor XRPD, Engineering Claire Pizzey SAXS Alex Dias MX Jitka Waterman MX Renjie Zhang XChem Sin-Yuen Chang XAS Jason van Rooyen Cryo-EM Sally Irvine Imaging

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

Biopharmaceutical Industry

Early Stage Drug Discovery Process

Beamlines & other facilities at Diamond

Macromolecular Crystallography Soft Condensed Matter Spectroscopy Materials Engineering and Environment Surfaces and Interfaces

I14

eBIC ePSIC

• 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)

• 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

• System compatible with external libraries
• Platform associated to I04-1 (optimized OAV

automated centering)

“In crystallo” screening of fragment libraries

Hits from JMJD2-DA (SGC)

Courtesy of Patrick Collins Diamond Light Source

Fragment Screening Platform (Xchem)

Soaking compounds Crystal harvesting Data collection Hit identification

15-30 m 1-2 days 1-2 days 1/2 day

Crystal volume ~5000x smaller than 100 micron ’standard’ I sec exposure - 2Å data

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

‘Class C GPCR metabotropic glutamate receptor 5 transmembrane domain.’ Doré, AS et al. Nature (2014) 511, 557-562

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) Main challenges

• Absorption
• Vacuum sample environment
• Analytical absorption correction

by X-ray tomography

• Large diffraction angles
• Curved detector assembly

P12M detector Novel in-vacuum vessel

Long Wavelength MX (I23)

Tomography camera Goniometer Sample position Fluorescence detector OAV

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

FORMULATRIX

Automatic sample handling 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)
• Serial crystallography

Electron Microscopy facility

I13 I14 and EM Facility Building Services Diamond

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

A National User Facility for Biological Electron Cryo-microscopy (eBIC)

• 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)

• BioSAXS sample changer
• In line HPLC and MALLS

Holbourn et al JBC (2011) 286 (25) 22243-22249

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

Parameters obtained by SAXS analysis

In Solution Protein Characterisation

B23 experimental room

Applications

• Protein stability and Kd determination
• HTCD - 96-well plates – compare protein

conformation under a range of conditions

• CD imaging of films - 2D mapping to assess

the homogeneity of the sample preparation

• High pressure SRCD – measure the secondary

structure content under high pressure

• Pump-probe SRCD for time-resolved studies

Rajasekaran et al, Biophys. Res. Com 398 (2010)

Circular Dichroism (B23)

• Spectroscopic technique to study a variety
• f chiral materials - small molecules

(drugs), polymers and biopolymers (nucleic acids, proteins, carbohydrates and lipids)

• Improved signal-to-noise ratio due to high

flux

Protein stabilisation upon binding of a metal ion

Infrared Spectroscopy (B22)

Lipid Protein Nucleic acid & Carbohydrate Water

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)
• 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

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

Nucleus

Nucleoli Nuclear membrane Mitochondria Nuclear pores ~ 120 nm in diameter

1 μm

Thank you

Jitka.waterman@diamond.ac.uk

http://www.diamond.ac.uk/industry.html

EUCALL Workshop: Biology at Advanced Laser Light Sources 30th November – 1st December 2017