Zn,Cu-SOD

HAH1

MT

GSH

Ctr1/2

CCS

Cu+

ATP7A/B

Mitochondrion

Cox17

D1D2

D3 D4D5

D6

Golgi complex

Cu+Zn,Cu-SODCCS

MTIMS

GSHSco2

Matrix

Cox11

Cytoplasm

CCO

Sco1

Cox172S-S

SecPr

SecPr

Welcome to the Session of the IG STRUCTURAL BIOLOGY!

The Infrastructural Scenario for Structural Biology

Lucia Banci Magnetic Resonance Center (CERM)

University of Florence

Structural Biology

Protein structure determination by NMR

In-cell NMR can monitor functional processes in live human cells

Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells.

Transfected HEK293T cells are used as a model system for human cells

Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution.

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.

HSSH

1H

15N

Cys 146

Cys 111

Cys 6

SH

Cys 57

1H

15N

apo-SOD1

E,Zn-SOD1apo-SOD1

In-cell NMR can monitor functional processes in live human cells

Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells.

Transfected HEK293T cells are used as a model system for human cells

Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution.

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.

+ Zn(II)

HSSHHSSH

SHHS

1H

15N

Cys 146

Cys 111

Cys 6

SH

Cys 57

1H

15N

E,Zn-SOD1

E,Zn-SOD1

In-cell NMR can monitor functional processes in live human cells

Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells.

Transfected HEK293T cells are used as a model system for human cells

Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution.

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.

SHSHCCS

SSS

S

+ Cu(I)

+ Zn(II)

HSSHHSSH

SHHS

1H

15N

Cys 146

Cys 111

Cys 6

SHS-S

Cys 57

1H

15N

Cu,Zn-SOD1

Cu,Zn-SOD1

Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013

HS SH

SHHS

HSSH

SHSH

SHSH

HS SH

SHHS

SHSH

HSHS

HSSH

SHHS

Zn(II)

S S

SS

SS

SS

HSHS

CCS

SHSHCCS

SSS

S

Cu(II)

Zn(II)Cu(I)

Following SOD1 maturation steps in

human cells

E,E-SOD1SH

E,Zn-SOD1SH

E,Zn-SOD1S-S

Cu(I),Zn-SOD1S-S

Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013

Structure of antigen fHbp

Heavy chain of monoclonal antibody

Mab502

Light chain of monoclonal antibody

Mab502

Fab region of

antibody

Structural Vaccinologya new approach in rational vaccine design

Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011

fHbp is very effective in inducing protective immunity eliciting antibodies but has different sequences and

different epitopes in different strains of MenB

antigen:antibody recognition

Structure-based design of a Vaccine against Mengingococcus B

Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011

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

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

INSTRUCT: The EU infrastructure for SB

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

Single crystal X-ray diffraction& NMR Single particle EM recons

Isolated particle EM tomography

Cellular EM tomography

Integrated Structural Biology – From single molecule to the cell

in vitro

in vivo

Cellular X-ray tomography / microscopy

And then …. modeling to put pieces together

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

Structural Genomics in the USA

Scal

e of

reso

lutio

n

Proteindomain

Proteincomplex

Virus

X-ray scattering

X-ray crystallography

Drosophila

mm

mm

nm

Bacteria

Eukaryotic Cells

X-ray imaging

EU I3 access to structural biology infrastructures and Instruct technology

The Bio-NMR ConsortiumProject Activities•Transnational Access (TA)

Activities•Joint Research Activities (JRA)•Networking Activities (NA)

19 Partners11 TA providers

Bio-NMR website www.bio-nmr.netBio-NMR Project Office: bionmr@cerm.unifi.it

Bio-NMR Project overview

EC FUNDING 9.0 million €

Beyond SB: metabolomics, ligand screening, in cell NMR, fibrillar aggregates, …

A unified access management system: from users’

applications to the evaluation of satisfaction after the visit

Stages of a TA application as monitored in the intranet

on-line submission Technical feasibility check by Local

Operators

Redirect to other facilityor

Ask for more information

Peer-review process by the International

Evaluation PanelRejection, if one declines

Acceptance with min. 2 positive votes

TA applicant

TA applicant receives an Intranet Account

LO notified, measurement slot allocation starts+

TA visit Visiting dates / machine time logged+

Notification/reminder e-mails to complete1. User data2. Publications3. Questionnaires

Protein structure determination by NMR

INSTRUCT was defined in the first ESFRI Roadmap

Preparatory Phase funded 2008 – 2011Transition Phase 2011 – 2013First Operations Phase 2013 – 2015

Rational for the construction of Instruct:

1.Vision of Integrated Structural Biology2.Increasing sophistication of technologies3.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

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

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 outcomesto initiate new technologies that will be the leading edge of an evolving,

globally competitive research infrastructure

INSTRUCT: The EU infrastructure for SB