Welcome to the Session of the IG STRUCTURAL BIOLOGY!

1. Welcome to the Session of the IG STRUCTURAL BIOLOGY! The Infrastructural Scenario for Structural Biology Lucia Banci Magnetic Resonance Center (CERM) University of Florence

2. Structural Biology

3. Protein structure determination by NMR

4. In-cell NMR can monitor functional processes in live human cells HS SH Understanding intracellular processes at the molecular level requires a high resolution description. In-cell NMR provides atomic-level information on a protein in the cellular environment. Transfected HEK293T cells are used as a model system for human cells apo-SOD1 15N Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells. Cys 146 15N SH Cys 57 Cys 111 1H Cys 6 Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution. E,Zn-SOD1 apo-SOD1 1H

5. In-cell NMR can monitor functional processes in live human cells SH HS HS SH SH HS Understanding intracellular processes at the molecular level requires a high resolution description. In-cell NMR provides atomic-level information on a protein in the cellular environment. Transfected HEK293T cells are used as a model system for human cells E,Zn-SOD1 15N Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells. + Zn(II) Cys 146 15N SH Cys 57 Cys 111 1H Cys 6 Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution. E,Zn-SOD1 1H

6. In-cell NMR can monitor functional processes in live human cells S S S S SH SH SH HS CCS HS SH SH HS Understanding intracellular processes at the molecular level requires a high resolution description. In-cell NMR provides atomic-level information on a protein in the cellular environment. Transfected HEK293T cells are used as a model system for human cells Cu,Zn-SOD1 15N Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells. + Zn(II) Cys 146 15N S-S SH Cys 57 Cys 111 1H Cys 6 Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution. Cu,Zn-SOD1 1H + Cu(I) Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013

7. Following SOD1 maturation steps in human cells SH SH SH S HS S SH SH HS SH S S HS HS S SH SH SH S HS S S S S S S SH HS HS SH SH SH CCS HS CCS HS SH SH HS HS Cu(I),Zn-SOD1S-S Zn(II) E,E-SOD1SH Cu(I) Zn(II) E,Zn-SOD1SH Cu(II) E,Zn-SOD1S-S Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013

8. Structural Vaccinology a new approach in rational vaccine design Structure of antigen fHbp Light chain of monoclonal antibody Mab502 Heavy chain of monoclonal antibody Mab502 fHbp is very effective in inducing protective immunity eliciting antibodies but has different sequences and different epitopes in different strains of MenB Fab region of antibody antigen:antibody recognition Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011

9. Structure-based design of a Vaccine against Mengingococcus B By knowing the structural properties of the antigens and of the epitopes in all the variants, a chimera antigen was produced which elicits complete protective immunity Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011

10. Small proteins Large proteins Multi-protein sub-cellular whole cells and domains and complexes assemblies structure INSTRUCT: The EU infrastructure for SB X-ray crystallography X-ray microscopy Light/fluorescent microscopy NMR mass spectrometry cryo-electron tomography small angle scattering single particle cryo-EM Computational analysis and modelling Core Centers: Oxford, Max Planck, CERM/CIRMMP, Weizmann, PSB Grenoble, IGMBC, EMBL A number of National and Regional facilities are affiliated to Instruct to promote Integrated Structural Biology in the country and in the region

11. Integrated Structural Biology – From single molecule to the cell Single crystal X-ray diffraction & NMR in vitro Single particle EM recons Isolated particle EM tomography Cellular EM tomography in vivo Cellular X-ray tomography / microscopy And then …. modeling to put pieces together

12. Structural Genomics in the USA SG has been funded within the Protein Structure Initiative (PSI) since 2000 The PSI program will be "sunset" in Jun 2015, as part of a general trend to sunset large team science and consortium projects in favor of R01 individual projects, possibly leading to less funding to SB as a whole

13. EU I3 access to structural biology infrastructures and Instruct technology mm Eukaryotic Cells Drosophila Bacteria Scale of resolution mm Virus Protein complex Protein domain X-ray imaging nm X-ray scattering X-ray crystallography

14. Bio-NMR website www.bio-nmr.net Bio-NMR Project Office: bionmr@cerm.unifi.it Bio-NMR Project overview The Bio-NMRConsortium Project Activities • Transnational Access (TA) Activities • Joint Research Activities (JRA) • Networking Activities (NA) EC FUNDING 9.0 million € A unified access management system: from users’ applications to the evaluation of satisfaction after the visit 19 Partners 11 TA providers Beyond SB: metabolomics, ligand screening, in cell NMR, fibrillar aggregates, …

15. Stages of a TA application as monitored in the intranet Technical feasibility check by Local Operators on-line submission Redirect to other facility or Ask for more information TA applicant Peer-review process by the International Evaluation Panel Rejection, if one declines Acceptance with min. 2 positive votes TA applicant receives an Intranet Account LO notified, measurement slot allocation starts + Notification/reminder e-mails to complete User data Publications Questionnaires Visiting dates / machine time logged TA visit +

17. Protein structure determination by NMR

18. INSTRUCT: The EU infrastructure for SB INSTRUCT was defined in the first ESFRI Roadmap Preparatory Phase funded 2008 – 2011 Transition Phase 2011 – 2013 First Operations Phase 2013 – 2015 Rational for the construction of Instruct: Vision of Integrated Structural Biology Increasing sophistication of technologies Alignment of national roadmaps – development of existing and new infrastructure, adoption of international standards Aim is to provide access to scientists across Europe to the best technology and training for structural cell biology

19. INSTRUCT: The EU infrastructure for SB Small proteins Large proteins Multi-protein sub-cellular whole cells and domains and complexes assemblies structure • to cover all resolution ranges using structural technologies • to exploit the power of using an integrated approach to address challenging problems and to achieve high impact research outcomes • to initiate new technologies that will be the leading edge of an evolving, • globally competitive research infrastructure X-ray crystallography X-ray microscopy Light/fluorescent microscopy NMR mass spectrometry cryo-electron tomography small angle scattering single particle cryo-EM Computational analysis and modelling