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
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. HS SH
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
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) HS SH
Cys 146
15N HS SH 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
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) HS SH
Cys 146
15N HS SH
S-S SH
Cys 57
Cys 111 1H
+ Cu(I) S
Cys 6 SH
CCS
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
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 cellsSH HS SH HS SH
Cu(I),Zn-SOD1S-S SH S HS SH S SH
CCS HS
CCS
Zn(II)
E,E-SOD1SH S
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
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
Structure of antigen
fHbp
Heavy chain of
monoclonal antibody
Mab502
Light chain of
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 9

10. 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
Small proteins Large proteins Multi-protein sub-cellular whole cells
and domains and complexes assemblies structure
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. X-ray crystallography
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 13

14. Bio-NMR Project overview
Project Activities
The Bio-NMR Consortium
Transnational Access (TA) Activities
Joint Research Activities (JRA)
Networking Activities (NA)
EC FUNDING 9.0 million €
Bio-NMR website
Bio-NMR Project Office:
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 s 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. Computational analysis and modelling
INSTRUCT: The EU infrastructure for SB
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
Small proteins Large proteins Multi-protein sub-cellular whole cells
and domains and complexes assemblies structure