Czech National Centre of the European Research Infrastructure Consortium INSTRUCT ERIC
A gateway to realm of structural data for biochemists, biophysicists, molecular biologist, and all scientists whose research benefits from accurate structure determination of biological macromolecules, assemblies, and complex molecular machineries at atomic resolution.
Open access to 10 high-end core facilities and assisted expertise in NMR, X-ray crystallography and crystallization, cryo-electron microscopy and tomography, biophysical characterization of biomolecular interaction, nanobiotechnology, proteomics and structural mass spectrometry.
A distributed infrastructure constituted by Core Facilities of CEITEC (Central European Institute of Technology), located in Brno, and BIOCEV (Biotechnology and Biomedicine Centre), located in Vestec near Prague, Central Bohemia.
6th European Crystallography School in Budapest
Registration to the 6th European Crystallography School to be held in Budapest, Hungary, between 5 and 11 July 2020 is now open.
The Instruct-ERIC Training Programme for 2020 is now online
Every year, Instruct-ERIC organises a programme of training events. These hands-on workshops cover a range of cutting-edge methods in structural biology to enable scientists to expand their expertise and implement new techniques in their research. Instruct training courses are delivered by world-renowned experts.
EU invests 10 million euro in unlocking technologies for key research in structural biology
To enable researchers from European institutes to extend innovative structural biology research, the EU has invested 10 million euro to iNEXT-Discovery, through its Horizon 2020 program.
Instruct-ERIC and ARBRE-MOBIEU Workshop: Analysis and Optimization of Sample Quality for Cryo-electron Microscopy and Other Structural Techniques
Workshop for structural biologists who want to improve their skills in sample analysis and optimisation for structural techniques, in particular Cryo-EM.
the best of science obtained using CIISB Core Facilities
J. Am. Chem. Soc. 2019
The unstructured C-terminal domain of delta subunit of bacterial RNA Polymerase is 90 aa long and highly charged. The charge distribution of this domain is distinct, with a conserved stretch of 9 residues (96−104) containing 7 positive charges followed by the rest of the domain with 51 acidic residues (K-D/E motif). A previous study demonstrated that the two parts of the motif transiently interact, and this affects the spatiotemporal properties of this domain. From the biological point of view, δ increases cell fitness and virulence of pathogens and was previously proposed to function as a nucleic acid mimic and affect RNAP− nucleic acid interactions.
Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in-vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.
Kuban, V., Srb, P., Stegnerova, H., Padrta, P., Zachrdla, M., Jasenakova, Z., Sanderova, H., Vitovska, D., Krasny, L..,Koval, T., Dohnalek, J., Ziemska-Legiecka, J., Grynberg, M., Jarnot, P., Gruca, A., Jensen, M.R., Blackledge, M., and Zidek, L.: Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase, J. Am. Chem. Soc. 2019, 141, 16817-16828, DOI:10.1021/jacs.9b07837
Science Advances 2019
Virion and genome organization of phage P68. (A and B) Structures of P68 virion, (C) genome release intermediate, and (D) empty particle. The whole P68 virion is shown in (A), whereas particles without the front half are shown in (B) to (D). The structures are colored to distinguish individual types of structural proteins and DNA. (E) Schematic diagram of P68 genome organization, with structural proteins color-coded in accordance with the structure diagrams shown in (A) to (D).
Phages infecting Staphylococcus aureus can be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect Gram-positive bacteria. Here, we present the structures of native S. aureus phage P68, genome ejection intermediate, and empty particle. The P68 head contains 72 subunits of inner core protein, 15 of which bind to and alter the structure of adjacent major capsid proteins and thus specify attachment sites for head fibers. Unlike in the previously studied phages, the head fibers of P68 enable its virion to position itself at the cell surface for genome delivery. The unique interaction of one end of P68 DNA with one of the 12 portal protein subunits is disrupted before the genome ejection. The inner core proteins are released together with the DNA and enable the translocation of phage genome across the bacterial membrane into the cytoplasm.
Hrebík, D., Štveráková, D., Škubník, K., Füzik, T., Pantůček, R., and Plevka, P.: Structure and genome ejection mechanism of Staphylococcus aureus phage P68, Sci. Adv. 2019, 5(10), eaaw7414, DOI: 10.1126/sciadv.aaw7414
literature to read, science to follow
In this section, a distinct selection of six highly stimulating research publications and reviews published during past 6 months is presented. It is our hope that links to exciting science, which deserves attention of the structural biology community, will help you to locate gems in the steadily expanding jungle of scientific literature. You are welcome to point out to any paper you found interesting by sending a link or citation to email@example.com. The section is being updated regularly.
Principles for Integrative Structural Biology Studies
Guru of integrative structural biology computation Andrej Sali and Michale P. Rout summarize in the recent Cell Primer Principles for Integrative Structural Biology Studies.
PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy
DNA–protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA–protein interactions at nanometer resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, Bart Hoogenboom, Alice Pyne et al. show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000–2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. They demonstrate – through the use of biotinylated DNA and streptavidin – that this selective adsorption extends to DNA–protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, they apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.
More than Proton Detection—New Avenues for NMR Spectroscopy of RNA
This minireview written by Harald Schwalbe, Boris Fürtig and coworkers reports on the development of NMR methods that utilize detection on low-g nuclei (heteronuclei like 13C or 15N with lower gyromagnetic ratio than 1H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum 1H coherences, detected by 13C NMR spectroscopy. Furthermore, 13C and 15N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-g nuclei detection experiments in RNA.
Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging
Cryo-electron tomography (cryo-ET) provides unprecedented insights into the molecular constituents of biological environments. In combination with an image processing method called subtomogram averaging (STA), detailed 3D structures of biological molecules can be obtained in large, irregular macromolecular assemblies or in situ, without the need for purification. The contextual meta-information these methods also provide, such as a protein’s location within its native environment, can then be combined with functional data. This allows the derivation of a detailed view on the physiological or pathological roles of proteins from the molecular to cellular level. Despite their tremendous potential in in situ structural biology, cryo-ET and STA have been restricted by methodological limitations, such as the low obtainable resolution. Exciting progress now allows one to reach unprecedented resolutions in situ, ranging in optimal cases beyond the nanometer barrier. Here, Florian Schur reviews current frontiers and future challenges in routinely determining high-resolution structures in in situ environments using cryo-ET and STA.
Chemical cross-linking with mass spectrometry: a tool for systems structural biology
Biological processes supporting life are orchestrated by a highly dynamic array of protein structures and interactions comprising the interactome. Defining the interactome, visualizing how structures and interactions change and function to support life is essential to improved understanding of fundamental molecular processes, but represents a challenge unmet by any single analytical technique. Chemical cross-linking with mass spectrometry provides identification of proximal amino acid residues within proteins and protein complexes, yielding low resolution structural information. This approach has predominantly been employed to provide structural insight on isolated protein complexes, and has been particularly useful for molecules that are recalcitrant to conventional structural biology studies. In this review, Juan D. Chavez and James E. Bruce discuss recent developments in cross-linking and mass spectrometry technologies that are providing large-scale or systems-level interactome data with successful applications to isolated organelles, cell lysates, virus particles, intact bacterial and mammalian cultured cells and tissue samples.
In‐Cell EPR: Progress towards Structural Studies Inside Cells
Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site‐directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in‐cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this minireview, the major advances of in‐cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.
Quote of January
“A journey of a thousand miles starts with a single step.”Laozi, 604 - 531 BC