The preface from the PhD Thesis:
‘Structural studies of regulatory protein domains involved in eukaryotic transcription’
by Olga Jasnovidova, 2017
________________________________________________________
‘Era of Integrative Structural Biology’
‘There is freedom waiting for you,
On the breezes of the sky,
And you ask "What if I fall?"
Oh but my darling,
What if you fly?’
© Erin Hanson
In 1976, the Protein Data Bank (PDB)
was launched containing 13 experimentally
determined structures. The number of
structures has rapidly increased since then,
reaching over 125,000 entries by the end of
2016 (www.pdb.org). The last 40 years can
thus be described as the ‘Era of Structural
Biology’.
Methods of structural biology have
become very powerful, providing valuable
mechanistic insights into basic biological
processes, which could be translated into
medical applications. Examples of such
knowledge include the structural models of
the DNA double helix, RNA polymerase,
spliceosome, ribosome, and nuclear pore
complex. The reconstitution of such and
other assemblies have led to increased
understanding of life processes.
The success of structural biology
methods depends heavily on the
technological advancements in
instrumentation, which have made it possible
to determine the structure of large molecular
assemblies and to increase the quality of the
structures determined for smaller molecules.
An excellent example is the development in
the field of cryo-electron microscopy
(CryoEM), which took place over the course
of the last few years1
.
Structural biology no longer represents
‘dark magic’ for many scientists, which is a
very important achievement. Nowadays
more than ever, nuclear magnetic resonance
(NMR), X-ray crystallography, and CryoEM
are more user-friendly, automated, and
accessible. Governments and private
funding bodies established large highthroughput
facilities that are accessible to a
broad range of users based on grant
application schemes. Therefore, from the
technical and financial point of view,
structure determination is easily accessible
for scientists from areas outside structural
biology. In fact, the inclusion and
consideration of structural data in biological
research has become an unspoken
requirement for publication in highly ranked
journals.
Structural genomics and other largescale
initiatives solved the structures of
many proteins and protein domains from
various genomes. It seems that all lowhanging
fruits have been picked, and the
discovery of novel folds is becoming a rare
event. Because of technological advances,
the level of complexity of the studied
biological systems has increased
significantly over the last years. In some
cases, the level of complexity is so high that
many phenomena cannot be addressed by
conventional structural biology methods
alone (NMR, X-ray crystallography, and
CryoEM). While conventional methods work
well for structured molecules, the presence
of flexibility, conformational changes, and
formation of transient complexes with weak
affinities remain the greatest challenge in
structural studies of macromolecules. An
additional limitation of contemporary
methods is related to the conditions under
which macromolecules are studied, involving
either concentrated solutions, crystals, or
vitrified ice, which differ significantly from the
conditions in the living cell. Current methods
fail to take into account the complexity of the
cell, the relevance of macromolecular
crowding, and the co-existence of other
phases such as aggregates, hydrogels, and
glasses. Therefore, the determination of
molecular structure directly inside the living
cell remains a challenge.
The scientific community has always
been aware of the above-mentioned
limitations, which has boosted the
development of many complementary
techniques. Consequently, the ‘Era of
Integrative Structural Biology’ became of
age, and this is a beautiful era2,3
.
What is the integrative approach, and
what does it offer us? For the purpose of this
preface, I would divide integrative structural
biology into two categories: first,
determination of a structural model by
integrating direct restraints from several
high- and low-resolution biophysical methods
2
; second, follow-up study of the determined
model, involving structure validation and
interpretation, using a complementary or
advanced variation of the original
investigative method.
Methods of the first category include
NMR, X-ray crystallography, CryoEM, smallangle
scattering, chemical crosslinking
followed by mass-spectrometry, and
computational approaches, which are used
to combine all the information. The individual
constraints gathered using different methods
provide restraints on the conformation,
position, and orientation of the components
in a macromolecular assembly. Combining
all restraints improves the accuracy,
precision, and completeness of a model,
especially when limited high-resolution
structural data on the entire assembly are
available2
.
The second category includes
experimental methods that are not directly
used for three-dimensional model
determination, but are invaluable for verifying
the model and characterizing its
behavior/dynamics. The verification of the
model includes determination of binding
affinity (e.g., by fluorescence anisotropy,
isothermal titration calorimetry, microscale
thermophoresis, electromobility shift assay),
confirmation of enzymatic activity by assays
specific for the studied system, and
validation of stoichiometry (e.g., analytical
ultracentrifugation, size exclusion
chromatography, small-angle scattering, light
scattering). Methods characterizing the
dynamics of the system can be very diverse
and usually include advanced biophysical
methods such as solution NMR
investigations (e.g., NMR relaxation
experiments, residual dipolar couplings), and
single-molecule methods (e.g., molecular
tweezers, single-molecule fluorescence
resonance energy transfer). Applying a
combination of methods allows moving from
the canonical, static understanding of the
structural model to a more dynamic
understanding of the process in action.
The availability of a wide range of
established complementary methods
indicates that we have reached an ‘Era
beyond the Technique’. Developments are
so fast and so broad that investigators are
no longer bound to a technique, but become
bound to the research question. In this
context, it might be no longer worth to define
ourselves by our training, e.g., structural
biologists, and it is rather necessary to train
ourselves in how to ask good questions and
how to communicate them to colleagues in
order to find solutions to these questions.
There are now powerful tools to aid us in our
quest. The future is bright.
Acknowledgement
I would like to thank R. Stefl, K. Kubicek, J.
Novacek, T. Shaikh, and C. M. Ionescu for the critical
reading of the text, discussion and suggestions.
References
1. Callaway, E. The revolution will not be crystallized:
a new method sweeps through structural biology.
Nat. News 525, 172 (2015).
2. Ward, A. B., Sali, A. & Wilson, I. A. Integrative
Structural Biology. Science 339, 913–915 (2013).
3. van den Bedem, H. & Fraser, J. S. Integrative,
dynamic structural biology at atomic resolution—
it’s about time. Nat. Methods 12, 307–318 (2015).