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The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association www.hecra.gr

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Page 1: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

The Power of Powder:

Protein Based Drug Screening

Irene Margiolaki University of Patras, Greece

& Hellenic Crystallographic Association

www.hecra.gr

Page 2: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

This document was presented at PPXRD -Pharmaceutical Powder X-ray Diffraction Symposium

Sponsored by The International Centre for Diffraction Data

This presentation is provided by the International Centre for Diffraction Data in cooperation with the authors and presenters of the PPXRD symposia for the express purpose of educating the scientific community.

All copyrights for the presentation are retained by the original authors.

The ICDD has received permission from the authors to post this material on our website and make the material available for viewing. Usage is restricted for the purposes of education and scientific research.

ICDD Website - www.icdd.comPPXRD Website – www.icdd.com/ppxrd

Page 3: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

STRATEGY

Page 4: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

WHO?

Page 5: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Method development

EU

UPAT

ESRF

PANalytical

NanoMEGAS

Novo Nordisk

NETWORK

Page 6: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

X-RAY CRYSTALLOGRAPHY / STRUCTURAL BIOLOGY

GROUP, DEPARTMENT OF BIOLOGY, UPAT

Fotini Karavassili (PhD) Alexandros Valmas (PhD)

Stavroula Fili (M.Sc.) Konstantina Magiouf (BSc.)

Danae Lachana (BSc.) Suzanna Logotheti (BSc.)

Stefanos Saslis (B.Sc.) Nikos Nikolopoulos (B.Sc.)

5

Our Team:

Page 7: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

6

Andy Fitch

Jon Wright

Yves Watier

European Synchrotron Radiation Facility ESRF, Grenoble, France

Page 8: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PANalytical

PANalytical, Almelo

Detlef Beckers

Thomas Degen

Celeste Reiss

Stjepan Prugovecki

Martijn Fransen

Gwilerm Nenert

UPAT, PATRAS

Stavroula Fili,

Partha Das (Former Scientist)

Fotini Karavassili

Alexandros Valmas

Konstantina Magiouf

Page 9: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Reviews

I. Margiolaki* & J. P. Wright*, Acta Cryst. A - Invited Review Article for Special Issue of IUCR[1]

• "Powder Crystallography on Macromolecules",

Acta Cryst. A64, 169-180 (2008) I. Margiolaki & J. P. Wright

• “Macromolecular Powder Diffraction”,

Book Chapter for the International Tables of Crystallography-

Volume H: Powder Diffraction, In Press I. Margiolaki

• “Macromolecular Powder Diffraction: Ready for genuine biological problems”

Protein & Peptide Letters, In Press F. Karavassili & I. Margiolaki

Page 10: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

What are the options?

NMR Spectroscopy X-ray Single Crystal

Diffraction

No need of crystals

Maximum MW limit

Crystals of ~ 50 - 100 μm

Crystallization may be problematic

Evans, G., Axford, D., Waterman, D., Owen, R.L.

Macromolecular microcrystallography.

Crystallography Reviews. 17, 105-142. 2011

Bieri M, Kwan AH, Mobli M, King GF, Mackay JP, Gooley PR.

Macromolecular NMR spectroscopy for the non-

spectroscopist: beyond macromolecular solution structure

determination.

FEBS J. 2011 Mar;278(5):704-15. 2011

Page 11: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Can we extract any structural information

from a protein polycrystalline sample?

Protein Crystalline Precipitates

Page 12: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

1980-1984: Micro-

crystals diffracting

to 30-40 Å (powder

patterns to 3.5 Å)

Preliminary powder data in the 80s

1985-1987

3 dimensional image

reconstruction by EM

at 25 Å showing the

internal ribosomal

tunnel

1986: excellent

diffraction, but

sever X-ray

damage:

Introducing cryo

bio- crystallography time-0 ; after 1 second Courtesy: Ada Yonath

Page 13: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

The first protein structure refinement using powder diffraction data:

Whale Metmyoglobin

R. B. Von Dreele

J. Appl. Cryst. (1999). 32, 1084-1089

Page 14: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Protein Crystalline Precipitates

Page 15: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

GOOD VS BAD PRECIPITATES

Courtesy: Prof. Terese Bergfors, http://xray.bmc.uu.se/~terese/tutorial2.html

Page 16: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Protein crystallization and phase diagrams: Neer Asherie, Methods 34 (2004) 266–272

Protein Crystallization & Phase Diagrams

Page 17: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PROTEIN POWDERS

Page 18: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association
Page 19: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

A VIRUS PROTEIN DOMAIN “SINGLE URCHIN” IN A GRID

http://www.mitegen.com/

Page 20: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

EMBL HC1b device

ID14- 2, PILATUS area detector λ=0.9934 Å Matthew Bowler, Yves Watier, Nicolas Papageorgiou

Papageorgiou et al., Z. Kristallogr. 225, 576–580 (2010)

Page 21: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PRESENTATION

• Data Collection

SR-XRPD + LAB XRPD

Page 22: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

http://www.spring8.or.jp/en/

1999 - present

ESRF- Grenoble, France

New collaborations with other synchrotrons

http://www.psi.ch/sls/ http://www.synchrotron-soleil.fr/

FRANCE JAPAN SWITZERLAND

http://www.spring8.or.jp/en/

Page 23: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

ID31: High Resolution Powder diffraction

Beamline

Detection of scattered beam using 9

APD detectors and 9 Si(111) analyser

crystals

Thanks to J.-L. Hodeau, M. Anne, P. Bordet, A. Prat, CNRS, Grenoble.

Fitch, J. Res. Natl. Inst. Stand. Technol. 109, 133-142 (2004)

Page 24: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

212019181716151413121110987654321

19,000

18,000

17,000

16,000

15,000

14,000

13,000

12,000

11,000

10,000

9,000

8,000

7,000

6,000

5,000

4,000

3,000

2,000

1,000

0

-1,000

-2,000

-3,000

-4,000

hkl_Phase 0.00 %

hkl_Phase 0.00 %

22212019181716151413121110987654

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

-200

hkl_Phase 0.00 %

hkl_Phase 0.00 %

2Th Degrees65.85.65.45.254.84.64.44.243.83.63.43.232.82.6

Inte

nsity (

Co

un

ts)

5,000

4,500

4,000

3,500

3,000

2,500

2,000

1,500

1,000

500

0

-500

hkl_Phase 0.00 %

ID31- λ= 1.300870(15) Å, LeBail fit in TOPAS

I222, a= 80.1888(5) Å, b= 96.2721(5) Å, c= 105.5448(5) Å

Sanofi Aventis

Uox complexed with its inhibitor 8-azaxanthine

0% PEG, pH 7.5

Margiolaki & Wright, Acta Cryst. A64, 169-180 (2008); Collings et al. Acta Cryst. D66, 539-548 (2010)

Page 25: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

LAB DIFFRACTOMETER

X’ PERT PRO BY PANALYTICAL

Page 26: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Insulin + nitrophenol (T3R3)

PAWLEY FIT OF LAB DATA

Valmas et al. Acta Cryst. (2015). D71, 819–828;

Novo Nordisk, Copenhagen

Page 27: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

POWDER DIFFRACTION:

A POWERFUL QUALITY CONTROL TOOL

FOR

PROTEIN- BASED DRUG SCREENING

Page 28: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

ASPECTS OF OUR STUDIES

▪ Polymorph Identification

▪ Structure solution of novel polymorphs

▪ Structure refinement

▪ Ligand identification using powder data

1

2

3

Page 29: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

UNIVERSITY - INDUSTRY COLLABORATION

Research Collaborators

Gerd Schluckebier & Mathias Norrman

Novo Nordisk, Copenhagen

Diabetes Protein Engineering

Page 30: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

THE CASE OF HUMAN INSULIN

Page 31: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

INSULIN HEXAMERIC CONFORMATIONS

• T6 B1-B8 EXTENDED CONFORMATION

• R6 B1-B8 HELICAL CONFORMATION

T3R3 3 MONOMERS B1-B3 EXTENDED

Β4-Β8 HELICAL

3 MONOMERS : Β1-Β8 EXTENDED

Page 32: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

• Several polymorphs exist depending on pH of crystallization and

concentration of additives.

• Great interest in finding new forms of potentially therapeutic applications.

• Study microcrystalline insulin crystallized as a function of pH (4 8.9) and

with phenol – based additives.

• Extremelly rich phase diagram.

MICROCRYSTALLINE INSULIN

Page 33: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

HUMAN INSULIN – LIGAND COMPLEXES

CRYSTALLINE PRECIPITATES

PHENOL RESORCINOL

Page 34: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

NEW

FORM C 2221

C 2 P 21

pH<5.6

Page 35: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

P21

a = 114.85 Å

b = 334.89 Å

c = 49.36 Å

= 103.36º

v = 1.85 million Å3

NEW POLYMORPH

pH 5.18 + resorcinol or phenol

Page 36: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

1.2x105

1.5x105

1.8x106

2.7x106

P21()C222

1P2

1()

Un

it C

ell

Vo

lum

e (Å

3)

pH

(b)

pH pH

phenol resorcinol

new

P 21

new

P 21

Karavassili et al., Acta Cryst. (2012). D68, 1632–1641

C2221 C2 P21

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

2.0x105

1.8x106

2.7x106

P21()

C2C2221 P2

1()

Un

it C

ell

Vo

lum

e (

Å3)

(a)

Page 37: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

HUMAN INSULIN – LIGAND COMPLEXES

CRYSTALLINE PRECIPITATES

M-cresol 4-nitrophenol

Page 38: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

HUMAN INSULIN – LIGAND COMPLEXES

CRYSTALLINE PRECIPITATES

M-cresol

4-nitrophenol

Low pH High pH

Page 39: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Valmas et al. Acta Cryst. (2015). D71, 819–828

ESRF- ID31

[λ = 1.29989 (3) Å, RT]

Page 40: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Valmas et al. Acta Cryst. (2015). D71, 819–828

HighScore Plus software

(Degen et al., Powder Diffr. 29, S13–S18

Page 41: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PHASE IDENTIFICATION

Valmas et al. Acta Cryst. (2015). D71, 819–828;

Page 42: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

RESULTS ON THE NEWS

http://mag.digitalpc.co.uk/fvx/iop/esrf/1503/

Page 43: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

ASPECTS OF OUR STUDIES

Polymorph Identification

▪ Crystal Screening

▪ Structure solution of novel polymorphs

▪ Structure refinement

▪ Ligand identification using powder data

2

Page 44: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PRESENTATION

• Structure Solution via MR

Page 45: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Doebbler, J. A. & Von Dreele, R. B.

Application of molecular replacement to protein powder data

from image plates.

Acta Cryst. D65, 348–355 (2009)

A NICE REVIEW ON MR

Page 46: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Collaboration with

EMBL – Hamburg

Mathias Wilmanns

Page 47: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

AN UNKNOWN PROTEIN STRUCTURE SOLVED FROM POWDERS

P212121, a= 24.70420(9) Å, b= 36.42638(14) Å,

c= 72.09804(26) Å

The Second SH3 domain of Ponsin

I. Margiolaki, J. P. Wright, M. Wilmanns, A. N. Fitch & N. Pinotsis

J. Am. Chem. Soc. 129, 11865-11871 (2007).

Q (Å-1)

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

Inte

nsity (

arb

itra

ry u

nits)

-1000

0

1000

2000

1.30 1.35 1.40 1.45 1.50 1.55 1.60

0

200

400

600

800

1000

Page 48: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Procedure followed for data analysis for SH3.2

Bhat, T. N. Calculation of an OMIT map. J. Appl. Cryst., 21, 279-281 (1988,)

Page 49: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association
Page 50: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

SH3.2: The final model

http://www.esrf.eu/news/general-old/general-2007/powder/

I. Margiolaki, J. P. Wright, M. Wilmanns, A. N. Fitch & N. Pinotsis. Second SH3 domain of ponsin solved from powder diffraction

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 129 (38): 11865-11871 SEP 26 2007

544 protein atoms and 36 water molecules were identified

in total OMIT and difference electron density maps.

Page 51: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PRESENTATION

• Structure Solution via MIR

Page 52: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

TEST SYSTEM

protein

Hen

egg-white

Lysozyme

(HEWL)

Molecular weight

Unit-cell (Å)

Space-group

14.4 kDa

a=b=79.2 c=38.0

P43212

Relative root mean

square intensity

change

(Crick and

Magdoff)

Gd (Z=64) : 0.40

Page 53: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

HEWL + Gd (Co-crystallisation)

Blue: Native

Red: +Gd

Wright et al., J. Appl. Cryst. 41, 329-339 (2008) & ESRF Scientific Highlights, p. 61-62 (2006).

Native HEWL to Gd derivative

Blue: Native

Red: +Gd

Page 54: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

THE SECONDARY STRUCTURE

• Using FFFEAR we are able to locate all the four helices of the lyzosyme

structure out of the five best fitted helices found by the program using the

standard fragment library.

Basso et al. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 7):756-61.

Page 55: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

THE HEWL MIR DENSITY MAP

Images created by Sebastian Basso

http://infoscience.epfl.ch/record/167174

Page 56: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

ASPECTS OF OUR STUDIES

▪ Polymorph Identification

▪ Crystal Screening

▪ Structure solution of novel polymorphs

▪ Structure refinement

▪ Ligand identification using powder data 3

Page 57: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

• COMBINED MULTIPLE PROFILES

• PERIODIC EXAMINATION OF TOTAL OMIT MAPS GENERATED

FOLLOWING BHAT’S PROCEDURE

• SYNERGY OF VARIOUS SOFTWARE PACKAGES

KEYPOINTS

Bhat, T. N. Calculation of an OMIT map. J. Appl. Cryst., 21, 279-281 (1988,)

I. Margiolaki & J. P. Wright, Acta Cryst. A64, 169-180 (2008)

Wright et al. Z. Kristallogr. Suppl. 26, 27-32 (2007)

Page 58: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

SOFTWARE USED

Powder Diffraction & CCP14 Single Crystal & CCP4 Home made

ESRF, APS

Area detector data integration

Fit2D

Indexing

DASH

Indexing & Pawley fit

HighScorePLUS

Data viewer

FULLPROF

Structure Rietveld refinement

GSAS

CCP4 software package

Molecular Replacement

MOLREP

PHASER

Structure Refinement

CNS

REFMAC

PHOENIX

Visualization Tools

WINCOOT

PYMOL

CHIMERA

Pawley fit

PRODD

Total Omit maps

SFCHECK

(modified version)

Data Extraction from

Synchrotron

Short routines in

PYTHON

Pycluster

ID31sum

Page 59: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PRESENTATION

Structure Refinement

The Flexible Rigid Body Approach in GSAS

Larson A. C. & Von Dreele R. B. (2004). General Structure Analysis System

(GSAS), Los Alamos National Laboratory Report LAUR 86-748.

Page 60: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

ENHANCED STRUCTURE REFINEMENTS

Rigid body description of amino acids

Margiolaki et al.

Acta Cryst. (2013). D69, 978–990

Page 61: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

Human Insulin microcrystals

co-crystallised with m-cresol

Optical Microscope Transmission Electron Microscope

THE KNOWN POLYMORPH R3 WITH ADDITIONAL LIGAND SITES

(R6 CONFORMATION)

Page 62: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

FRB RIETVELD ANALYSIS:

8 COMBINED SR-XRPD AND LAB- XRPD PROFILES

Page 63: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

FITTING OF MODEL TO OMIT MAP

62

Page 64: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

THE KNOWN POLYMORPH R3 WITH ADDITIONAL LIGAND SITES

(R6 CONFORMATION)

Karavassili et al., In Preparation

Page 65: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

PRESENTATION

• Future & Concluding Remarks

Page 66: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

FUTURE

• CRYSTAL SCREENING

Polymorph Identification & Ligand Binding

Crystal size and morphology (XFEL)

Phase Mapping

Page 67: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

FUTURE

• COMBINED USE WITH

XFEL measurements on nano-crystalline precipitates

Electron Diffraction on single nano-crystals

• Spence, Weierstall, Chapman , Rep. Prog. Phys. 75, 102601, 2012

Barty, Küpper, Chapman , Annual Review of Physical Chemistry 01/2013

• Three-dimensional electron crystallography of protein microcrystals

Shi et al., 2013, eLIFE

Page 68: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

UPAT, Greece

S. Fili

F. Karavasili

A. Valmas

D. Lahana

S. Logotheti

S. Salsis

N. Nikolopoulos

Former members:

P. P. Das

A. Stewart

K. Magiouf

A. E. Giannopoulou

M. Kalatha

E. Kotsiliti

ESRF, Grenoble

Andy Fitch

Jon Wright

Yves Watier

The ID31 team

Former members :

Lucy Saunders

Ines Collings

Sotonye Dagogo

Lisa Knight

Mark Jenner

Sebastian Basso

ACKNOWLEDGMENTS

X-RAY DIFFRACTION

Page 69: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

COLLABORATORS

EMBL, Hamburg

Nikos Pinotsis

Matthias Wilmanns

APS, Chicago

Bob Von Dreele

Soleil, Paris

Gavin Fox

EPFL, Lausanne

Marc Schiltz

Celine Besnard

CINaM, Marseille

Marion Giffard

Françoise Bonneté

AFMB, Marseille

Bruno Canard

Nicolas Papageorgiou

Bruno Coutard

Violaine Lantez

Novo Nordisk, Copenhagen

Gerd Schluckebier

Mathias Norrman

PaNalytical, Netherlands

Detlef Beckers

Thomas Degen

Celeste Reiss

Stjepan Prugovecki

Martijn Fransen

University of Manchester

John Helliwell

University of Amsterdam

Henk Schenk

University of

Geneva

Radovan Cerny

Nanomegas

Stavros Nicolopoulos

Bruker, Germany

Diederik Ellerbroek,

Cees Baas

Patrick Romijn

Robbert Jan Brandenburg Sanofi Aventis, Montpellier

Mohamed El Hajji

Bertrand Castro

Excelsus Structural

Solutions SPRL

Fabia Gozzo

Page 70: The Power of Powder: Protein Based Drug Screening · The Power of Powder: Protein Based Drug Screening Irene Margiolaki University of Patras, Greece & Hellenic Crystallographic Association

IAEA

Coordinated Research Project (CRP) on

“Utilisation of accelerator-based real-time

and in-situ methods in investigation of

materials for energy applications”

2012-2015

CRP code: F12024

UNESCO & L’OREAL Foundations

International

Fellowship for Women in Science

2010-2012

Nanomegas

Stavros Nicolopoulos

2012-Present

EU & University of Patras

• FP7: SEE-DRUG

PI: George Spyroulias

http://www.seedrug.upatras.gr/

2012-2015

• Aristeia II

PI: Irene Margiolaki

2014-2015

• COST

PI: Fraser McMillan

2014-2018

FUNDING