Czech National Centre of the European Research Infrastructure Consortium INSTRUCT ERIC

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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.

CIISB Core Facilities Research Highlights 

T. O. Koller, et al.: The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition, J. Amer. Chem. Soc. (2023), 145, 851-863 https://doi.org/10.1021/jacs.2c08816

D. Farci, et al.: The structured organization of Deinococcus radiodurans’ cell envelope, PNAS (2022) e112101, https://doi.org/10.1073/pnas.2209111119

M. Genova, et al.: Tubulin polyglutamylation differentially regulates microtubule-interacting proteins, EMBO Journal, (2023) e112101, https://doi.org/10.15252/embj.2022112101

A. Schenkmayerova, et al.: Catalytic mechanism for Renilla-type luciferases, Nat. Catal. (2023). https://doi.org/10.1038/s41929-022-00895-z

M. Stiborek, et al.: Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging Anal. Chem. (2022) 94, 51, 18114 – 18121, https://doi.org/10.1021/acs.analchem.2c05216

More publications

CIISB Research Highlights

the best of science obtained using CIISB Core Facilities

  • Nature 2023

    Nature 2023

    Overview of the dormant ribosome structure from 1 hpf zebrafish (a) and Xenopus egg (b). Ribosome-associated factors are shown as surface representations; eEF2b and eIF5a correspond to Protein Data Bank (PDB) accessions 6MTE and 5DAT, respectively.

    Andrea Pauli Research Group

    Significance

    Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4–eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

    Leesch, F. et. al.: A molecular network of conserved factors keeps ribosomes dormant in the egg, Nature (2023), 613, 712-720: https://doi.org/10.1038/s41586-022-05623-y

  • JACS 2022

    JACS 2022

    Interaction of Myxovalargin with the E. coli ribosome. (a) Relative position of P-site tRNA (green) and erythromycin (Ery, cyan) to 23S rRNA nucleotides protected from DMS by 10 μM (light blue) or 100 μM (dark blue) MyxB.

    Andreas Kirschning, Daniel N. Wilson, and R. Müller Research Groups

    Significance

    Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

    Koller, T.O., Scheid, U., et al.: The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition, J. Amer. Chem. Soc. (2023), 145, 851-863 https://doi.org/10.1021/jacs.2c08816

  • PNAS 2022

    PNAS 2022

    Features comparison between T4P-like structure obtained by cryo-EC, remote homology detection, and subtomogram averaging. The main dimensions of the T4P-like complex obtained by 3D cryo-EC (Center Left; EMD-14097), remote homology detection (Center), and subtomogram averaging (Center Right; EMD-14096) are compared. The Left and Right boxes show comparable images of the slice through at equivalent levels, from Top to Bottom, for the T4P-like complex obtained by cryo-EC (left box) and by cryo-ET subtomogram averaging (right box), both computed imposing a p6 symmetry. The dark-yellow boxes indicate the noncrystalline regions that are missing in the model obtained by cryo-EC with respect to the model obtained by subtomogram averaging. The S-layer (SL), the outer membrane (OM), and the inner membrane (IM) thicknesses are indicated. Scale bars indicate 50 Å.

    Dario Piano Research Group

    Significance

    The cell envelope of the extremophile bacterium Deinococcus radiodurans was studied by cryo-electron microscopy and described with unprecedented detail. In this bacterium, the outermost cell envelope layer, named surface layer, is characterized by a highly regular tiling of proteins extending their crystalline organization to the cell envelope layers below (until the inner membrane). The study shows three main protein complexes, with masses in the MDa range, regularly organized into an astonishing geometrical regularity. The observed organization contributes to protecting the cell against environmental stressors and maintaining an efficient permeation of environmental solutes.

    Surface layers (S-layers) are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by cryo-electron microscopy, finding the crystalline regularity of the S-layer extended into the layers below (outer membrane, periplasm, and inner membrane). The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity yet allowing a certain degree of free space. The presented results suggest how S-layers, at least in some species, are mesoscale assemblies behaving as structural and functional scaffolds essential for the entire cell envelope.

    Farci, D., Haniewicz, P., and Piano, D.: The structured organization of Deinococcus radiodurans’ cell envelope, PNAS (2022) e112101, https://doi.org/10.1073/pnas.2209111119

More publications Research Highlights archive

Reader’s Corner

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 readerscorner@ciisb.org. The section is being updated regularly.


 

9 Feb

Structure-based design of bitopic ligands for the mu-opioid receptor (Nature)

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site found in µOR and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gisubtypes and show strongly reduced arrestin recruitment—one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Reader’s Corner Archive

Quote of March 

“The opposite of a correct statement is a false statement. But the opposite of a profound truth may well be another profound truth.”

Niels Bohr

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