CIISB 2020 - A Short Outlook into the Foreseeable Future in 6654 Characters
A revolution in the resolution of structural data obtained through cryo-electron microscopy, seen during last decade, brought about a change in the fundamental paradigms of structural biology. Mapping of the 3D spatial arrangement of the basic components of cellular systems with atomic resolution, still reliant on their crystallization and study by X-ray diffraction or measurement in solution by nuclear magnetic resonance, has acquired a new tool that bridges the limitations of the two methodologies. The development of cryo-electron microscopy applications has been documented by an exponential increase in the number of laboratories equipped with the most powerful microscopes over the past three years and by massive investments in this technology not only in Europe but also in Asia and the United States. Cryo-electron microscopy allows the reconstruction of 3D structures in an environment of amorphous ice that preserves the native character of the measured samples. The development of single-particle reconstruction methods, accelerating rapidly over the last twenty years, was rewarded in 2017 with the Nobel Prize in Chemistry, shared by Jacques Dubochet, Joachim Frank and Richard Henderson. Current and anticipated developments in electron microscopy make it possible to determine the structures of macromolecules and their complexes with a resolution better than 2Å, which will allow the use of cryo-EM structures for drug development. Classification of macromolecules seen in an electron microscope opens up possibilities to describe the dynamics of macromolecules, which is highly important for understanding their biological function and which has been difficult to study.
The progress in cryo-electron microscopy has strongly stimulated the development of cryo-electron tomography. This technique shifts structural biology from the field of in-vitro studies, whether extracted from cell cultures or prepared by recombinant techniques, to the field of in-vivo study in the cell environment. Tomography complements studies of three-dimensional macromolecular structures by allowing their localization in a cellular environment. Understanding where, when, and how biomolecules work in a cell is critical to uncovering the underlying mechanisms in cell biology. Recent developments in fluorescent light microscopy allow the protein to be displayed in living cells. However, because of the labeling used, fluorescence microscopy can only display selected biomolecules or organelles, but not the structural context in the cell. Correlative light and electron microscopy (CLEM) allows the localization of specific or time-dependent cell processes by fluorescence labeling with subsequent high-resolution analysis by cryo-electron microscopy. It can be expected that continued development will revolutionize and standardize the correlative light and electron microscopy and will allow its full inclusion in the standard arsenal of methods for studies of cellular molecular processes.
The development of a method for determining macromolecular structures through electron diffraction on micro-crystals also has great potential. This approach combines the use of very small crystals on which structural data can be obtained using available electron microscopes, minimizing thus necessity to use the synchrotron sources of X-ray radiation even for difficult projects. Electron diffraction has the potential to combine the ease and flexibility of measurement in an electron microscope with computationally efficient and rapid determination of macromolecular structures through diffraction on a crystal.
Other integrative structural biology technologies are also undergoing major methodological developments. Serial femtosecond crystallography, using high-intensity coherent pulses of laser radiation from XFEL (X-ray Free Electron Laser) sources, enables the efficient collection of diffraction data from micro- and nanocrystals, allowing the study of the time evolution and dynamics of biomolecular structures with femtosecond time resolution. The development of new NMR spectroscopy methods in high fields has made it possible to detect and structurally characterize so-called invisible, minor, or transient states of biomacromolecules that are elusive for other methods. In parallel to the development of new technologies (direct detectors for electron microscopy, high-temperature superconductivity materials for the design of NMR magnet spectrometers with a working frequency of 1.1 and 1.2 GHz, the launch of the European XFEL source in Hamburg), computational methods are also evolving rapidly to integrate the results obtained by various techniques. The rapid development is also evidenced by the fact that the PDB (Protein Data Bank) has created a separate PDB-Dev archive to store these "integrative" structures. Research in integrative structural biology supports the study of general principles governing cellular processes. New knowledge gained in this field of science contributes significantly not only to the expansion of basic general knowledge, but also speeds up and facilitates applied research in the development of new medicines.
CIISB will strive to maintain cutting-edge technology and knowledge in key fields of structural biology. Expected technology developments are reflected in the CIISB Development Strategy, which was already prepared for the implementation of downstream operations and investment support projects, and which envisages the necessary recovery and reinvestment steps in line with the developments described above. At the same time, the emphasis will be on training and educating the staff of the CIISB core facilities so that they can implement the new methodologies on an ongoing basis to fully exploit the potential of the newly procured equipment.
The CIISB infrastructure is ready to adapt the capacity and staffing of its laboratories to the needs of technology using integration into international networks in terms of resources and by recruiting relevant experts and by training the existing staff.
The focus will continue to be on the quality of users' outputs, predominantly in international scientific journals with high impact.
The rollout of an online form of an open-access application and project management is also planned for the period from 2020 onwards, which will greatly facilitate the process for evaluation and implementation of the requested measurements and services.
While the CIISB infrastructure is secured in terms of operation and investments through two newly obtained grants of OP VVV CIISB UP (CZ.02.1.01/0.0/0.0/18_046/0015974) and a dedicated support project (LM2018127) for years 2020-2022, efforts will continue to support mainly development activities through new national and international grant projects.
We perceive with rising urgency the need to provide available expertise and laboratory capacity to prepare samples for structurally-biological studies. The diverse range of techniques available in CIISB and the natural limitations of users in terms of understanding the techniques and their particular demands mean that more challenging projects can only be implemented after lengthy optimization of user samples. The demands on sample quality often start at the expression and purification of target molecules or complexes alone. The CIISB Molecular Structure Center (Biocev IBT) therefore plans a pilot operation of the central protein expression laboratory after evaluating the community interest.
Given the importance of emerging electron techniques in structural biology and the long-term underfunding of this area at the BIOCEV center, CMS CIISB also plans developments in the direction of electron-based techniques (diffraction and microscopy).