natural innovators

user group meeting

21st

– 22nd

April 2015

The Talking Point Conference Centre

TTP Labtech Head Office

Melbourn Science Park, Melbourn, UK

natural innovators

Programme

Tuesday 21st April

08.30 Registration and coffee

09.00 Welcome

09.15 Joerg Benz – Roche

Protein crystallisation for structure based drug design

09.45

Shilong Fan - Structural Biology Centre, Tsinghua University

The future of Chinese structure biology and why we need to set up this

platform for the Chinese biology scientist

10.15

Stephane Boivin – EMBL

Shining light with Mosquito-LCP at the EMBL crystallization facility - European

Synchrotron Radiation PETRA-III

10.45 Break

11.00

Andrew Doré – Heptares Therapeutics Ltd

Crystal Structure of a Class C GPCR – Metabotropic Glutamate receptor 5

(mGluR5)

11.30

Maria Håkansson – SARomics Biostructures

mosquito applications for crystallography and liquid dispensing for biophysics

at SARomics Biostructures

12.00 Lunch

13.00 David Hargreaves – AstraZeneca

A million mosquito tips

13.30

Marieke Lamers – Charles Rivers Laboratories

HDAC 4 crystallography to support inhibitor design as a potential therapy for

Huntington’s disease

14.00

Gebhard F.X. Schertler – Paul Scherrer Institut

Lipidic cubic phase crystallisation - from an exotic idea to a very important

method in the structural biology of membrane proteins

14.30 Break

14.45 Development team presentations and blue sky discussions

15.45 Site tour

17.00 Return coach to Cambridge

17.45 Trinity canapé reception

18.10 Cambridge & Trinity College tour

19.30 Trinity dinner

21.30 Coach back to Quy Mill and Holiday Inn hotels

natural innovators

Wednesday 22nd April

08.30 Coffee

09.00 Isabel Moraes – MPL, Diamond Light Source

High-throughput membrane protein structure determination

09.30 Daniel Picot – Institut de Biologie Physico-Chemique

dragonfly and the rethinking of membrane proteins crystallisation

10.00 Break

10.15

Alexey Rak – Sanofi-Aventis

The benefits gained from using a mosquito assisted high throughput

microseeding technique

10.45 Final questions and round table discussion

12.15 Lunch

13.15 Coach back to Quy Mill and Holiday Inn hotels

natural innovators

Meet the European liquid handling team:

natural innovators

Joerg Benz PhD, Senior Principal Scientist, Roche Pharmaceutical Research

and Early Development, Roche Innovation Center, Basel, Switzerland

Joerg obtained his PhD in 1996 from the Max-Planck-Institute of

Biochemistry in Martinsried in the group of Robert Huber studying

structure and function of peripheral membrane proteins. After an

additional year as postdoc at the Max-Planck-Institute working on

kinases, he joined the group of Ulrich Baumann at the University of

Berne in Switzerland where he was involved in the structural

characterization of initiation events of eukaryotic translation.

In 2001, Joerg decided to leave academia and continue his career in

the pharmaceutical industry. Currently, he is heading the protein

crystallisation facility in the department of Molecular Design and Chemical Biology at Roche

Pharmaceutical Research and Early Development (pRED), Basel.

Protein crystallisation for structure based drug design

High resolution 3D X-ray structures of drug targets in the presence or absence of small

molecule ligands are a requirement for structure-based drug design. High quality crystals are

a must for the method but the production of high quality crystals is very often still a major

bottleneck in the drug-design cycle. In the presentation we will provide an overview how we

try to overcome the problem of high quality crystal generation at Roche. Major emphasis will

be given to examples for which seeding, limited in situ proteolysis and generation of several

crystal forms were important, both for soluble proteins and membrane proteins.

natural innovators

Shilong Fan PhD, X-ray facility manager, Structural Biology Centre, Tsinghua

University, Beijing, China

Shilong gained his PhD in biochemistry and biophysics in 2008 from

the University of Science and Technology of China. After that, he

joined Dr. Ermanno Gherardi’s group at the Oncology department,

Cambridge University to continue structural biology research for three

years.

In 2012, Shilong returned to China and joined the Structural Biology

Centre, Tsinghua University, as X-ray facility manager.

In the last two years, Shilong has worked on estabilshing an

automated macromolecule structural biology service platform. This

includes facility service and technology service for Tsinghua University.

The future of Chinese structure biology and why we need to set up this

platform for the Chinese biology scientist

natural innovators

Stephane Biovin PhD, Staff Scientist, Sample Preparation and Characterisation

Facility, European Molecular Biology Laboratory, Hamburg, Germany

Stephane Boivin obtained in 2006 his PhD from and international joint

program in chemistry from the National Scientific Research Institute

(Canada) and in biology from the University of Rouen (France). His

research projects consisted to characterise binding site of vasoactive

hormone that acts through a G-protein-coupled receptor and design

structural analogs. Following his PhD he undertook a first postdoctoral

position at the University of Texas Health Science Center at Houston

(USA).

Responsible for the crystallisation and homology modelling platform,

his main project concisted to develop and characterize catalytic antibodies (abzymes) as

vaccines to prevent or treat AIDS and Alzheimer’s disease. Stephane joined EMBL Grenoble

Oustation for his second postdoctoral position where he worked to understand the

adaptation of avian influenza A virus to the human host in a pandemic context. Since 2010,

Stephane moved to EMBL-Hamburg as staff Scientist to be in charge of the SPC facility and

high-throughput crystallisation platform.

Shining light with mosquito-LCP at the EMBL crystallisation facility - PETRA3

synchrotron

The integrated Sample Preparation and Characterisation (SPC) facility is located next to the

PETRA beamlines. It consists in a full-scale facility equipped with state-of-the-art equipment

to carry out purification and characterisation of macromolecular samples that are destined

for X-ray crystallography (MX) or small angle scattering (SAXS). In addition to the biophysics

platform, users can take advantage of our high-throughput crystallisation platform which

meet the challenges of both standard and new cutting-edge experiments for soluble and

membrane proteins, as lipid-cubic phase. Our ultimate goal is to create a pipeline from

bench to beamline. I will be presenting how mosquito-LCP play a key role in serving a mixed

community of local EMBL scientists, beamline visitors and scientists from the European

Union research area. In addition, I will present an overview of new developments for which

mosquito-LCP instrument and TTP Labtech IQ plate represent a masterpieces.

natural innovators

Andy S. Doré PhD, Associate Director Crystallography, Heptares Therapeutics

Ltd., Welwyn Garden City, UK

Andy directs the crystallography group at Heptares Therapeutics

overseeing all crystallographic projects undertaken by the company.

He established the crystallography group and laboratories, including

protocols for cross platform GPCR crystallisation, runs all synchrotron

trips and his group has solved over 70 GPCR crystal structures to

date, including class A and the inaugural class B and class C receptor

structures. Prior to setting up the crystallography section at Heptares

in December 2009, he was a postdoctoral training fellow in the group

of Professor Laurence Pearl (FRS) at the Institute of Cancer Research

in London for 5 years. Andy completed his DPhil in Biochemistry and Protein Crystallography

in the laboratory of Professor Sir Tom Blundell (FRS) at the University of Cambridge in 2004

having previously worked for New England BioLabs in Boston, MA as an International

Visiting Research Scholar. His first degree specialised in Molecular Biology and Genetics.

Crystal structure of a class C GPCR – metabotropic glutamate receptor 5

(mGluR5)

natural innovators

Maria Håkansson PhD, Principal Scientist, SARomics Biostructures AB, MAX IV

Laboratory Crystallisation Facility Manager, Lund, Sweden

Maria Håkansson obtained her PhD in Molecular Biophysics in 2001

from Lund University. The thesis was focused on X-ray crystallography

and was called ”Structural stuides of metal-binding proteins revealing

dynamic behaviour”. Following her PhD she took the opportunity to

learn molecular biology and protein expression as a postdoc at the

Clinical Chemistry Department at Malmö University Hospital, Sweden.

From 2006 and onwards Maria has been working for SARomics

Biostructures. She has also helped to set up the crystallisation facility

at the MAX IV Laboratory synchrotron. She has successfully worked

with crystallisation, structure determination and biophysical characterisation of many

different proteins and protein-ligand complexes.

mosquito applications for crystallography and liquid dispensing for

biophysics at SARomics Biostructures

Standard and not so standard applications of the mosquito from the daily workflow at

SARomics Biostructures will be described. Among the standard applications is crystallisation

of protein/ligand complexes ranging from small fragments to protein–protein complexes.

Seeding and additive screening belong to the common techniques employed. However we

also use the mosquito in a non-standard way as a liquid handling robot enabling easy mixing

of samples for biophysical characterisation such as dynamic light scattering and differential

scanning fluorimetry (Thermofluor).

natural innovators

David Hargreaves PhD, Associate Principal Scientist, Discovery Sciences,

Structure & Biophysics, AstraZeneca, Cambridge, UK

David studied Biochemistry and Microbiology as a mature student at

the University of Sheffield after which he went on to gain a PhD in X-

ray Crystallography in Prof. David Rice’s lab. David’s thesis work

resulted in the first visualisation of the DNA Holliday Junction bound to

its partner protein RuvA. David then completed 5 years post doctoral

study on DNA helicases and plasmid maintenance systems after

which he joined AstraZeneca.

David supports small molecule drug discovery projects across

oncology working with groups in Boston and Sweden and is leading a

long running project looking at the utility of antibodies and other macromolecular entities as

tools in crystallisation.

A million mosquito tips

The mosquito has been the core piece of equipment in our High Throughput Crystallography

facilities both here in the UK, Sweden and Boston. In this talk I will describe my experiences

with it over the last 10 years which will include how we operate our standard screening

protocol and some of the more esoteric uses of the Mosquito both in crystallography and

other disciplines.

natural innovators

Marieke Lamers PhD, Principle Scientist, BioFocus Discovery Services, Charles

River Laboratories, Saffron Walden, UK

Marieke joined BioFocus in September 2008 and as a group leader of

the BioFocus structural biology team has responsibility for

management of the group. In this role she is involved in the execution

and overseeing of projects concerning construct design, cloning,

expression, purification and crystallisation to enable successful protein

structure determination.

Previously Marieke was one of the founding members of Sareum Ltd, a

Structure-Based Drug Discovery Company. As Head of Protein

Sciences she was responsible for the co-ordination of the protein

expression, purification and crystallisation for both internal and external structural biology

programs. Through ten years of working in a contract research organisation, firstly with

Sareum and then with BioFocus, Marieke has interacted with a diverse customer base

including large pharmaceutical businesses and small biotech companies on a variety of

discovery research projects.

HDAC 4 crystallography to support inhibitor design as a potential therapy for

Huntington’s disease

HDAC4 crystallography at CRL was part of an integrated program in collaboration with the

CHDI Foundation, Inc. (Los Angeles, USA), to develop catalytic-site small molecule

inhibitors of the Class IIa histone deacetylases HDAC4, 5, 7 and 9. The CHDI Foundation, a

biomedical research foundation devoted to discovering Huntington’s disease-modifying

therapies, collaborated with CRL, to crystallise HDAC4 in a state amenable to drug design.

Inhibition of Class IIa HDAC enzymes have been suggested as a therapeutic strategy for a

number of diseases, including cancer, muscle wasting disorders, Huntington’s disease and

various metabolic disorders. This optimization process to develop catalytic-site small

molecule inhibitors was supported by structural biology, exemplified by the co-crystallization

of selected inhibitors with the catalytic domain of human HDAC4.

Initially the structures of wild type HDAC4 in complex with three different CHDI inhibitor

molecules were solved, but analysis of the results were complicated by the presence of

close crystal packing interactions at the active site. Subsequently, a program to develop a

more robust structural model for HDAC4 crystallography was undertaken, and this resulted

in development of a mutant enzyme crystal structure with much improved crystal packing,

allowing reliable study of enzyme ligand interaction. Through this process the mosquito

Crystal was used for primary screen set-ups and optimization studies which were crucial in

obtaining well-diffracting crystals.

natural innovators

Prof Gebhard Schertler, Head of Biology and Chemistry Department, Paul

Scherrer Institut, Villigen PSI, Switzerland

Prof. Gebhard Schertler is investigating the structure and function of G

protein coupled receptors (GPCRs). When the group was located at

the MRC Laboratory for Molecular Biology (Cambridge, UK), they

focused on 3D structural analysis by X-ray and electron

crystallography and solved the atomic structures of several GPCRs

(rhodopsin, beta adrenergic receptors). With this expertise, Schertler

successfully revealed the mechanisms of light-induced rhodopsin

activation and agonist-binding to a GPCR.

Since his move to the Paul Scherrer Institute in 2009, Schertler has established an

interdisciplinary research group on GPCRs, including crystallography, electron microscopy,

NMR, biophysics and bioinformatics and a platform for protein

expression/purification/crystallization. He is also responsible for biological applications on

the Swiss Free Electron Laser (SwissFEL) and is involved in the experimental setup and

design of biology beamlines for optimization of both biomolecular nanocrystallography and

biological X-FEL imaging. Schertler is leading the Biology and Chemistry Department and is

a member of the Board of Directors at the Paul Scherrer Institute (PSI). He is also a

Professor for Structural Biology at the ETH in Zürich, Switzerland.

Lipidic cubic phase crystallisation - from an exotic idea to a very important

method in the structural biology of membrane proteins

The origin of lipidic cubic phase (LCP) crystallisation was the ingenious meeting of structural

biology with the study of lipids and lipidic substances. Rosenbusch and Landau brought

together two fields of research and created a novel crystallisation method that can fulfill at

once the optimisation of hydrophobic and hydrophilic interactions. They also were able to

develop reliable protocols to execute the experiments, and with the crystallisation of bacterial

rhodopsin with LCP they had a true breakthrough. It took many efforts from many people to

get to more automated and material saving procedures and finally first robotic automation

was possible. The mosquito LCP robot was not the first LCP robot but it was an attempt to

develop a dedicated platform optimised for the high through-put laboratory. It was based on

a prototype built by Schertler’s team at the MRC Laboratory of Molecular Biology.

natural innovators

Isabel Moraes PhD, Membrane Protein Laboratory group leader, Imperial

College/Diamond Light Source, Didcot, UK

Since 2010 Isabel has led the Membrane Protein Laboratory (MPL) at

Diamond Light Source. At the MPL, she is responsible for the

management of the facility as well as its scientific research. Isabel

provides scientific advice and guidance to all projects making use of

the MPL.

She is also responsible for the establishment of internal and external

collaborations (national and international). She has established

collaborations with the Diamond MX beamline scientists regarding to

the development of new methodologies in crystallisation and structure

determination of membrane proteins.

In the last few years she has organised many courses and workshops in field of membrane

proteins in the UK and abroad. Previously to MPL, Isabel worked in many years as a

structural biologist in industry where she gained vast experience in drug discovery. Her PhD

degree and postdoctoral position were also in structural biology applied to drug discovery

projects in collaboration with industry. She enjoys working in multi-disciplinary enviroments

and being involved in scientific public events. Recently, she has been involved in the use of

X-ray Free Electron Lasers (X-FEL) in the field of membrane protein structural biology.

High-throughput membrane protein structure determination

It is estimated that nearly 30% of proteins encoded in the human genome are membrane

proteins. They perform a variety of functions including the transport of nutrients and ions,

transport of water into and out of cells, removal of waste products and toxins from cells

produce, and the use oxygen in respiration and photosynthesis. About 60% of the

commercially available drugs target membrane proteins. Therefore, the study of membrane

protein structures provides a basic understanding of life at the molecular level and helps in

the rational and targeted design of new drugs, which could reduce unwanted side effects.

The advent of the genomics and proteomics initiatives combined with high-throughput

technologies such as automation, miniaturization, integration, and third-generation

synchrotrons have enhanced membrane protein structure determination rate. Yet, many are

the obstacles a membrane protein structural biologist researcher has to face before reaching

its final destination, the 3D structure!. I will present the latest strategies regarding to the

production of suitable crystals in membrane protein structure determination alongside with

latest strategies used at synchrotron beamlines regarding to the screening and data

collection of such demanding crystals.

natural innovators

Daniel Picot, CNRS Research Director, Institut de Biologie Physico-Chimique

(IBPC), Paris, France

Daniel Picot is a researcher in the Laboratory of Physical Chemistry of

Membrane Protein at the IBPC. After a PhD at the Biozentrum Basel,

he joined the group of Michael Garavito at the University of Chicago,

one of the earliest groups solely devoted to membrane protein

crystallography. He participated in early struggles there to crystallise

membrane protein and solved the structure of prostaglandin H

synthase.

In Paris, he is working on an integrated approach to study electron

transfer chains of bioenergetics membranes. He determined the

structure of the complex cytochrome b6f of oxygenic photosynthesis. His work has given him

the opportunity of tackling various crystallisation approaches and new surfactants. He

currently hopes to grasp supramolecular membrane assemblies with the help of

crystallography. At the IBPC, he has also set up and coordinated the crystallographic facility.

dragonfly and the rethinking of membrane proteins crystallisation

Crystallisation of membrane protein makes use of a large spectrum of approaches:

crystallisation in or ex micellar phases, lipidic cubic phases, sponge phases, lamellar

phases, etc. Those utilise a broad range of surfactants and cosurfactants. This nearly infinite

space search has led to a grim outlook for membrane protein crystallography that could be

only overcome by the development of very high throughput strategies. Nevertheless, it has

been recognised since the earliest day of membrane protein crystallisation that surfactants

play an important role, for example through phase transition processes. These theoretical

considerations are in agreement with early or recent survey on crystallisation condition.

Could instrumentation like the dragonfly helps to have a more rational approach? I will try to

confront earlier pre-dragonfly observations with our recently acquired instrument.

natural innovators

Alexey Rak PhD, Head of Structural Biology (LGCR), Sanofi-Aventis R&D,

Vitry sur Seine, France

Dr. Alexey Rak, Ph.D.-biochemistry and biophysics, is heading the

structural biology and biophysics efforts at the LGCR (Lead Generation

to Candidate Realization) scientific platform of Sanofi R&D. He

obtained his M.Sc in biology and genetics and second M.Sc in

Biochemistry, followed by PhDs in Biochemistry and Biophysics.

He was awarded with Prize of The Association for the Advancement of

Biomedical Sciences in 2003, European Young Investigator Award in

2004 and Peter und Traudl Engelhorn Foundation research prize in

2005 for his work in the field of vesicular membrane trafficking and

small GTPases associated signaling that he conducted at Max-Planck Institute for Molecular

Physiology in Dortmund, Germany. In 2007 Dr. Rak joined Sanofi, a leading pharmaceutical

company based in Paris, France, and is now leading structural biology and biophysics based

research in small molecules and biologics therapeutic projects for various human diseases

indications at Sanofi R&D in France.

The benefits gained from using a mosquito assisted high throughput

microseeding technique

Protein crystallisation is a major bottleneck for protein structure determination, and it still

remains the least standardised part of the protein structure determination process by X-ray

crystallography. Nucleation is the first and critical step to succeed with protein crystallisation.

Crystal nuclei (seeds) can be transferred from drop to drop to increase success with the 3D

growth of crystals. This seeding technique was introduced very early and was already

mentioned in 1979 by Blundell and Johnson[1]. This method revolutionised the protein

crystallisation process.

Microseed Matrix Seeding (MMS) was introduced by C.G. Ireton and B. Stoddard [2]. Allan

D’Arcy and co-workers expanded this method by automating the procedure and seeding

directly into crystallisation screens [3]. The method has been successfully demonstrated to

be applicable for general use and has showed success on different classes of proteins in

generating new space groups, improving diffraction quality, and finding useful hits when

there were none before [4].

TTP Labtech’s mosquito instrument is found to be essential for MMS application in high

trough put industrial operations at Sanofi’s labs. mosquito’s applications for MMS

crystallisation will be discussed during the presentation.

[1]T.L. Blundell, L.N. Jonson (1976) Protein Crystallography, Academic Press, Science [2] C.G. Ireton and B. Stoddard (2004) Microseed matrix screening to improve crystals of yeast cytosine deaminase. Acta Cryst. D60, 601-605 [3] D'Arcy A, Villard F, Marsh M. (2007) An automated microseed matrix-screening method for protein crystallization. Acta Cryst. D63, 550-4. 2007 [4] D'Arcy A, Bergfors T, Cowan-Jacob SW, Marsh M.(2014) Microseed matrix screening for optimization in protein crystallization: what have we learned? Acta Crystallogr F Struct Biol Commun. 2014 Sep;70(Pt 9):1117-26

natural innovators

Useful information

Attendee list

Alexey Rak Sanofi-Aventis

Aline Reynaud EPFL

Allan Surgenor Vernalis R&D

Andrew Doré Heptares Therapeutics Ltd

Annette Schousboe Petersen Novo Nordisk A/S

Artem Muravev Qvadros-Bio

Ayaka Take AsOne

Colin Levy University of Manchester

Daniel Picot Institut de Biologie Physico-Chemique

David Hargreaves AstraZeneca

Dean Derbyshire Medivir AB

Desley Pitcher AXT

Dimitri Chirgadze University of Cambridge

Dirk Gewert Horizon Discovery

Fabrice Gorrec MRC, Cambridge

Gebhard Schertler Paul Scherrer Institut

Ilka Mueller Biofocus Discovery Services - Charles Rivers Laboratories

Isabel Moraes MPL, Diamond Light Source

Joerg Benz Roche

Jorge Enrique Gonzalez Reyes Gonzalez Reyes & Cia

Julie Mosley Glaxosmithkline

Keishi Nakayama AsOne

Kor Kalk Rug

Kornelius Zeth Universidad del País Vasco/Euskal Herriko Unibertsitatea

Linda Oster AstraZeneca

Margarete Neu Glaxosmithkline

Maria Hakansson SARomics Biostructures AB

Marieke Lamers Biofocus Discovery Services - Charles Rivers Laboratories

Masahiro Matsushita AsOne

May Marsh Paul Scherrer Institut

Mindy Tan BioLab

Rafael Couňago Structural Genomics Consortium

Sarah Schulze UCB S.A. - Celltech

Sarveshwar Johri TTP Labtech India

Shilong Fan Tsinghua University

Shou Lee CSBiotech

Stephane Boivin European Molecular Biology Laboratory

Xiao Feng LBD

Ying Yang LBD

natural innovators

Internet connection

Wifi name: TTP-Guest password: talkingpoint

Coach arrangements

Coaches have been arranged each day to pick up and drop off UGM guests between the

two recommended hotels (Cambridge Quy Mill hotel and Holiday Inn Lake View hotel) and

TTP Labtech.

Tuesday 21st

7.30 am pick up from Holiday Inn Lake View, 7.45 am pick up from the Cambridge

Quy Mill hotel

5pm pick up from the TalkingPoint Conference Centre to Cambridge tour and dinner

9.30/10pm return journey back to the hotels

Wednesday 22nd

7.30 am pick up from Holiday Inn Lake View, 7.45 am pick up from the Cambridge

Quy Mill hotel

1.15pm return journey back to the hotels

Parking in Cambridge

If you would like to join the Cambridge tour and Trinity College dinner but prefer to drive

in/out of Cambridge, the nearest recommended car park can be found in the Grand Arcade:

https://www.cambridge.gov.uk/car-parks-map. Please speak to the UGM coordinator for

further details on where to meet the group.

First aid assistance

First aiders can be found by dialing ‘0’ on any phone.

In the event of a fire

If you find a fire, please quickly inform a member of TTP Labtech as soon as possible, do not

attempt to put it out yourself.

In the event of a fire please make your way quickly and calmly to the fire meeting point near

the park entrance where your presence will be noted by the UGM coordinator.

natural innovators

Notes

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

natural innovators

Notes

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

....................................................................................................................

natural innovators