Welcome to the Session of the IG
Cytoplasm
STRUCTURAL
BIOLOGY!
MT
Zn,Cu-SOD
GSH
Mitochondrion
The Infrastructural Scenario for
IMS
Cu
Cu
Structural Biology
CCS
CCS
+
+
Cox11
MT
Ctr1/2
HAH1 Cox17
Lucia Banci
D1
Zn,Cu-SOD
CCO
Cox172S-S
Matrix
Sco1
Sco2
GSH
D6
Magnetic
Resonance
Center (CERM)
D5
D3
ATP7A/B
UniversityD4 of FlorenceGolgi complex SecPr
D2
SecPr
Structural Biology
Protein structure determination by NMR
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.
Cys 146
HSSH
15N
SH
Cys 57
1H
Maturation processes such as
protein folding, post translational
modifications (i.e. metal binding,
disulfide bond formation) are
followed at atomic resolution.
Cys 6
E,Zn-SOD1
apo-SOD1
1H
Cys 111
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)
Cys 146
HSSH
HSSH
SH
HSSH
Cys 57
1H
Maturation processes such as
protein folding, post translational
modifications (i.e. metal binding,
disulfide bond formation) are
followed at atomic resolution.
15N
Cys 6
E,Zn-SOD1
1H
Cys 111
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)
HSSH
S-S
HSSH
SH
Cys 57
1H
15N
Cys 146
HSSH
Cys 111
+ Cu(I)
SH
SH
Maturation processes such as
protein folding, post translational
modifications (i.e. metal binding,
disulfide bond formation) are
followed at atomic resolution.
S
S
Cys 6
S
S
Cu,Zn-SOD1
1H
Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013
Following SOD1
maturation steps in
human cells
HS SH
Cu(I),Zn-SOD1S-S
SH
SH
SH
HS
HS SH
HS SH
SH
HS
SH
SH
HS SH
S S
S
S
HS SH
HS
HS
E,E-SOD1SH
SH
SH
SH
SH
SS
CCS
Zn(II)
HS
HS
Cu(I)
Zn(II)
E,Zn-SOD1SH
S
S
S
S
S
S
Cu(II)
E,Zn-SOD1S-S
Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013
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
Structure of antigen
fHbp
Light chain of
monoclonal antibody
Mab502
Heavy chain of
monoclonal antibody
Mab502
Fab region
of
antibody
antigen:antibody
recognition
Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011
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
INSTRUCT: The EU infrastructure for SB
Small proteins
and domains
Large proteins Multi-protein
and complexes assemblies
X-ray crystallography
NMR
sub-cellular
structure
whole cells
X-ray microscopy
Light/fluorescent microscopy
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
Integrated Structural Biology – From single
molecule to the cell
in vitro
Single crystal X-ray diffraction
& NMR
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
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
Scale of resolution
mm
Eukaryotic
Cells
Bacteria
mm
Virus
Protein
complex
Protein
domain
nm
X-ray imaging
X-ray scattering
X-ray crystallography
Drosophila
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 www.bio-nmr.net
Bio-NMR Project Office: bionmr@cerm.unifi.it
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, …
Stages of a TA application as monitored in the intranet
on-line submission
TA applicant
Technical feasibility
check by Local
Operators
Redirect to other facility
or
Ask for more information
Peer-review process
by the International
Evaluation Panel
Rejection, if one declines
Acceptance with min. 2 positive votes
TA applicant receives an
Intranet Account
Notification/reminder e-mails
to complete
1.User data
2.Publications
3.Questionnaires
TA visit
+
LO notified, measurement
slot allocation starts
+
Visiting dates /
machine time logged
Protein structure determination by NMR
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:
1.Vision of Integrated Structural Biology
2.Increasing sophistication of technologies
3.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
 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
Small proteins Large proteins Multi-protein sub-cellular
and domains and complexes assemblies
structure
whole cells
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