All CIISB Core facilities are fully functional. Visits of external foreign users are regulated by the Measures concerning foreigners and border crossing of the Czech Government. Please, check the current status on the web and contact the staff for details.
CIISB offers priority access to groups that need to use CIISB structural biology services for projects directly related to studies of the virus and projects aiming to develop an effective vaccine or treatment. To request priority access, please submit a research proposal with „COVID-19“ in the title of the proposal, through the online application system HERE. Successfully accepted proposals will be free of charge, and no financial contribution will be requested for the measurement/service.
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.
CEITEC Core Facilities
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Webinar series: What can iNEXT-Discovery do for us? Armin Wagner, DIAMOND, Long-wavelength crystallography – Experiments on the edge
The webinar series What can iNEXT-Discovery do for us? will continue on November 30 at 16:00 CEST with the topic Long-wavelength crystallography – Experiments on the edge presented by Armin Wagner, DIAMOND.
[Crystals, IF 2.404] Paper Invitation for Special Issue “Advances in Protein Crystallization and Crystallography “
You are cordially invited to contribute a research article, review, short communication or perspective to a Special Issue of Crystals journal called “Advances in Protein Crystallization and Crystallography “.
Open Research Europe - The European Commission open access publishing platform
European commission is preparing the launch of Open Research Europe, the European Commission scientific publishing service, which will provide Horizon 2020 and Horizon Europe beneficiaries with a venue to publish their results in full compliance with open access policies.
Highlights of Coronavirus Structural Studies
Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes (Cell Reports)
Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, Shapiro, L., Kwong, P.D. et. al. designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from similar to 0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. They also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors (Science)
Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. D. Baker et.al. designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concentration (IC50) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC50 similar to 0.16 nanograms per milliliter). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.
Advanced methods in macromolecular crystallization IX
Course, originally planned for May 2020, is postponed to May 9-15 2021.
The main aim of this course is consideration of theoretical aspects of crystal growth process as well as practical work: there will be a healthy mix of advanced discussions of the theory and of laboratory experiments and the possibility for participants to crystallize their own proteins.
the best of science obtained using CIISB Core Facilities
(−)-Bactobolin A and selected related natural products.
(A) Overall structure of ovine complex I. Core subunits necessary for the reaction of complex I are labeled with corresponding colors, and mammalian supernumerary subunits are shown in gray. NADH and quinone binding sites are indicated. The membrane arm contains four separate proton-pumping channels: three in the antiporter-like subunits ND2, ND4, and ND5 and one in the E-channel, composed of subunits ND1, ND6, and ND4L. Q, quinone.
Complex I is the first and, with 45 subunits and a total mass of ~1 MDa, the most elaborate of the mitochondrial electron transfer chain enzymes. Complex I converts energy stored in chemical bonds into a proton gradient across the membrane that drives the synthesis of adenosine triphosphate (ATP), the universal energy currency of the cell. In each catalytic cycle, the transfer of two electrons from nicotinamide adenine dinucleotide (NADH) to a hydrophobic electron carrier quinone, which happens in the peripheral arm of the enzyme, is coupled to the translocation of four protons across the inner mitochondrial membrane in the membrane arm. The exact mechanism of this energy conversion currently presents an enigma because of complex I’s size and the spatial separation between the two reactions.
To understand the coupling mechanism of complex I, we solved its cryo–electron microscopy (cryo-EM) structures in five different conditions, including the substrate- and inhibitor-bound states and during active turnover, unlocking the various conformations that the enzyme goes through during the catalytic cycle. We also improved the resolution to up to 2.3 to 2.5 Å, allowing us to directly observe water molecules critical for proton pumping.
Kampjut, D.and Sazanov, L.A. The coupling mechanism of mammalian respiratory complex I
Science, 2020, 370, eabc4209, https://doi.org/10.1126/science.abc4209
Angew. Chem. Int. Edit. 2020
The participation of reactants undergoing a polarity inversion along a multicomponent reaction, allows the continuation of the transformation with productive domino processes. Thus, indole aldehydes in Groebke-Blackburn-Bienaymé reactions lead to an initial adduct which spontaneously triggers a series of events leading to the discovery of novel reaction pathways together with a direct access to a variety of linked, fused and bridged polyheterocyclic scaffolds. Indole 3- and 4-carbaldehydes with suitable isocyanides and aminoazines afford fused adducts through oxidative Pictet-Spengler processes, whereas indole 2-carbaldehyde yields linked indolocarbazoles under mild conditions, and a bridged macrocycle at high temperature. These novel structures are potent activators of the human aryl hydrocarbon receptor signaling pathway.
Ghashghaei, O., Pedrola, M., Seghetti, F., Martin, V.V., Zavarce, R., Babiak, M., Novacek, J., Hartung, F., Rolfes, K.M., Haarmann-Stemmann, T., and Lavilla, R.: Extended Multicomponent Reactions with Indole Aldehydes: Access to Unprecedented Polyheterocyclic Scaffolds, Ligands of the Aryl Hydrocarbon Receptor Angew. Chem. Int. Ed. on line https://doi.org/10.1002/anie.202011253
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.
Structure of inhibitor-bound mammalian complex I (Nat. Commun.)
Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, V.R.I. Kaila, J. Hirst et. al. describe the 3.0-Ao resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. They combine the structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Their findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I. The respiratory complex I (NADH:ubiquinone oxidoreductase) is a large redox-driven proton pump that initiates respiration in mitochondria. Here, the authors present the 3.0 angstrom cryo-EM structure of complex I from mouse heart mitochondria with the ubiquinone-analogue inhibitor piericidin A bound in the active site and with kinetic measurements and MD simulations they further show that this inhibitor acts competitively against the native ubiquinone-10 substrate.
Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry (Nat. Commun.)
Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, H. Urlaub et. al. present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Their approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 they show that the technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei, they determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that their workflow is not restricted to reconstituted materials. As the approach can distinguish between protein-RNA and DNA interactions in one single experiment, they project that it will be possible to obtain insights into chromatin and its regulation in the future. Cross-linking mass spectrometry (XLMS) allows mapping of protein-protein and protein-RNA interactions, but the analysis of protein-DNA complexes remains challenging. Here, the authors develop a UV light-based XLMS workflow to determine protein-DNA interfaces in reconstituted chromatin and isolated nuclei.
Sensitivity enhancement of homonuclear multidimensional NMR correlations for labile sites in proteins, polysaccharides, and nucleic acids (Nat. Commun.)
Multidimensional TOCSY and NOESY are central experiments in chemical and biophysical NMR. Limited efficiencies are an intrinsic downside of these methods, particularly when targeting labile sites. This study reported by L. Frydman et. al. demonstrates that the decoherence imparted on these protons through solvent exchanges can, when suitably manipulated, lead to dramatic sensitivity gains per unit time in the acquisition of these experiments. To achieve this, a priori selected frequencies are encoded according to Hadamard recipes, while concurrently subject to looped selective inversion or selective saturation procedures. Suitable processing then leads to protein, oligosaccharide and nucleic acid cross-peak enhancements of ≈200–1000% per scan, in measurements that are ≈10-fold faster than conventional counterparts. The extent of these gains will depend on the solvent exchange and relaxation rates of the targeted sites; these gains also benefit considerably from the spectral resolution provided by ultrahigh fields, as corroborated by NMR experiments at 600 MHz and 1 GHz. The mechanisms underlying these experiments’ enhanced efficiencies are analyzed on the basis of three-way polarization transfer interplays between the water, labile and non-labile protons, and the experimental results are rationalized using both analytical and numerical derivations. Limitations as well as further extensions of the proposed methods, are also discussed.
Cryo-EM analysis of a membrane protein embedded in the liposome (PNAS)
Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here Fan, X., Yan, N. et.al. present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 angstrom resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Their approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
Retrieving functional pathways of biomolecules from single-particle snapshots (Nat. Commun.)
A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, des Georges, Singharoy, Frank, and Ourmazd et. al. present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Their approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations. There is a great interest in retrieving functional pathways from cryo-EM single-particle data. Here, the authors present an approach that combines cryo-EM with advanced data-analytical methods and molecular dynamics simulations to reveal the functional pathways traversed on experimentally derived energy landscapes using the ryanodine receptor type 1 as an example.
Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing (Nat. Commun.)
Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well- studied heat-sensing receptor that is also activated by vanilloid compounds, including cap- saicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here W.D.Van Horn e.al. show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.
Quote of November
“Wisdom is not a product of schooling but of the lifelong attempt to acquire it.”Albert Einstein