BIOLOGIA STRUTTURALE

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1 LUCI DI SINCROTRONE Presente e futuro delle grandi sorgenti di raggi X e UV italiane nel contesto europeo: BIOLOGIA STRUTTURALE Martino Bolognesi Dept. BioSciences University of Milano, Italy

2 Biological Crystallography vs. Structural Biology

3 Structural Biology MX, SAXS, WAXS NMR EM NS MS AFM & advanced microscopy BIO techniques: Sample preparation, Biochemistry, Biophysics, Cell Biology, Spectroscopies,

4 X-ray diffraction based investigations (MX) Overall throughput: PDB, post-sg, SR sources Achievements: > 6 Nobel Prizes Aims: mechanisms, drug design, basic principles High reaching targets: complexes, membrane proteins, micro/nano-crystals Future of X-ray sources

5 Current PDB holdings and database growth MX

6 Current PDB holdings and database growth - NMR

7 Current PDB holdings and database growth - EM

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9 X-ray diffraction based investigations (MX) Overall throughput: PDB, post-sg, SR sources Achievements: > 6 Nobel Prizes Aims: mechanisms, drug design, basic principles High reaching targets: complexes, membrane proteins, micro/nano-crystals Future of X-ray sources

10 Nobel Laureates linked to MX Perutz & Kendrew, 1962 for Hb and Mb Hodgkin, 1964 for VitB12... Deisenhofer, Michel & Huber, 1988 for photosynthetic reaction center Mac Kinnon, 2003 for K + channels Ramakrishnan, Steitz, Yonat, 2009 for Ribosome Lefkowitz & Kobilka, 2012 for G prot.coupl.recept. (...Lipscomb (1976), Klug (1982), Hauptam & Karle (1985), Walker (1997), Kornberg (2006)...)

11 Myoglobin 16 kda Ribosome 2.5 MDa

12 X-ray diffraction based investigations (MX) Overall throughput: PDB, post-sg, SR sources Achievements: > 6 Nobel Prizes Aims: mechanisms, drug design, basic principles High reaching targets: complexes, membrane proteins, micro/nano-crystals Future of X-ray sources

13 Targeting viral disease Effective treatments are lacking for many important diseases caused by viruses: influenza, SARS, hepatitis, yellow fever, dengue,...

14 Crystal structure of hnv RdRp bound to NF023

15 Mastrangelo et al. J.Mol.Biol. (2012)

16 Drug-Lead Molecular Fragments primary binding site secondary binding site Croci et al. PlosOne (2014)

17 CC 25 nm 12.5 nm 6.3 nm Yellow Fever Virus infected cell cultures (incr. Ivermectin conc.) 3.1 nm 1.5 nm 0.7 nm VC E BEE EC patent A novel therapeutic use of an active ingredient belonging to the class of Ivermectins

18 X-ray diffraction based investigations (MX) Overall throughput: PDB, post-sg, SR sources Achievements: > 6 Nobel Prizes Aims: mechanisms, drug design, basic principles High reaching targets: complexes, membrane proteins, micro/nano-crystals Future of X-ray sources

19 PROTEIN COMPLEX STRUCTURES (IN EPIGENETICS)

20 Transcription factors in chromatin structure/assembly Nardini et al. CELL (2013)

21 NF-YB and NF-YC electrostatic interactions to DNA Nardini et al. CELL (2013)

22 Conserved protein/dna recognition rules

23 X-ray (RT) data collection in crystallization trays De Colibus et al. Nat.Struc.Mol.Biol. (2014)

24 MEMBRANE PROTEINS

25 G-protein coupled receptors (GPCR) cover about 15% of the druggable human genome (30% of market) Haga et al. Nature (2012) Ring et al. Nature (2013) Shukla et al. Nature (2013)

26 A VIEW INTO THE NEAR FUTURE Nano-crystal diffraction on X-FEL sources (serial fs crystallography) Diffract_before_explosion paradox: X-FEL may overcome MX hurdles, i.e. crystal size and radiation damage Single molecule diffraction Time-resolved diffraction Successful cases

27 Demirci et al. Acta Cryst F69 (2013)

28 Lysozyme micro/nano-crystals diffraction pattern Barends et al. Nature (2014)

29 Barends et al. Nature (2014)

30 Successful ab initio SAD phasing Barends et al. Nature (2014)

31 Cathepsin G in cell grown crystals were used for X-FEL based diffraction Redecke et al. SCIENCE (2013)

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33 Conclusions Access to synchrotron sources will remain in high demand, in parallel with increasing X-FEL access Source access programs will be crucial Multidisciplinary training and views needed International policy for MX needed

34 Dipartimento di Bioscienze Universita di Milano 34