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
BIOCEV Core Facilities
Instruct-ERIC Webinar Series: Structure Meets Function - Webinar #3
Instruct-ERIC webinar series continues on October 13 and will highlight some of the latest developments in structural biology, demonstrating how integrative methods are enabling scientists to decipher the mechanisms that underpin health and disease. Talks will be given by Bert Janssen, Markus Weingarth and Meindert Lamers.
Instruct-ERIC Best Practices in Cryo-EM Workshop
University od Leeds, United Kingdom, is organising an online training on the best practises in Cryo-EM. The workshop will be held on 12-13 October 2020.
Sessions will include presentations on best practice in sample preparation, imaging and data handling/processing, operational models in light of COVID-19, as well as round table discussions focused on specific topics.
A Celebration of Instruct-ERIC: Achievements, Impact and Expanding Ambitions
It is our great pleasure to invite you to a virtual event celebrating the achievements of Instruct-ERIC in the time since achieving European Research Infrastructure Consortium (ERIC) status in 2017. The event will celebrate the achievements of Instruct-ERIC, recognising the hard work and commitment of the consortium, staff, supporters and users, in the wider context of the successful European Research Infrastructure landscape.
Highlights of Coronavirus Structural Studies
Growth, detection, quantification, and inactivation of SARS-CoV-2 (Virology)
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. M.S. Diamond et. al. have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.
SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation (Nat. Struct. Mol. Biol.)
The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, O. Mühlemann, N. Ban et.al. show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. They observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in human cells. Based on the high-resolution structure of the 40S– Nsp1 complex, they identify residues of Nsp1 crucial for mediating translation inhibition. They further show that the full-length 5′ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.
Instruct-ERIC Best Practices in Cryo-EM Workshop
The workshop is intended to allow those involved in the running of high-end cryoEM facilities to discuss and share best practice, and is open to EM facility scientists, manager and computing specialists both from academia and industry.
the best of science obtained using CIISB Core Facilities
Nature Communications 2020
Schematic illustration of the TRAK1-mediated anchoring of KIF5B. a Top: in absence of TRAK1, KIF5B (green) can either continue its walk by rebinding the disengaged motor domain to the microtubule or dissociate from the microtubule when the engaged motor domain unbinds from the microtubule. Bottom: in presence of microtubule-bound TRAK1 (magenta), when both motor domains of KIF5B disengage from the microtubule, KIF5B remains tethered to the microtubule through a diffusive interaction of TRAK1 with the microtubule and thereby enables the rebinding of a motor domain of KIF5B to the microtubule. In this state, TRAK1 might facilitate navigation around obstacles by diffusion along the microtubule surface. b Overview of the functions of TRAK1. Top: TRAK1 activates auto-inhibited KIF5B, enabling its processive movement along microtubules. Middle: TRAK1 increases the processivity of KIF5B in crowded environments. Bottom: TRAK1 enables KIF5B-based transport of isolated mitochondria along microtubules in vitro.
Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin- 1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, pro- viding an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.
Henrichs, V., Grycova, L., Bařinka, C., Nahačka, Z., Neužil, J., Diez, S., Rohlena, J., Braun, M., and Lánský, Z.:Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments, Nat. Commun. (2020) 11, 3123, https://doi.org/10.1038/s41467-020-16972-5
Nature Communications 2020
Sinefungin recognition by the nsp16 MTase. A) SARS CoV-2 nsp10-nsp16 protein complex bound to sinefungin (white sticks), nsp16 in surface representation (cyan), nsp10 in cartoon representation (orange) and zinc ions as gray spheres. B) Detailed view of sinefungin recognition, important amino acid residues are shown in stick representation, water as red spheres and hydrogen bonds are shown as dashed lines.
COVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2'-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.
Krafčíková, P., Šilhan, J., Nencka, R., and Bouřa, E.: Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation, Nat. Commun. (2020) 11, 3717, https://doi.org/10.1038/s41467-020-17495-9
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 firstname.lastname@example.org. The section is being updated regularly.
Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET (J.Amer.Chem.Soc.)
Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. G-N.W. Gomes and C. Gradinaru et.al. used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, they examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, they integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Their results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.
In-cell architecture of an actively transcribing-translating expressome (Science)
Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. J. Rappsilber et. al. combined whole-cell cross-linking mass spectrometry, cellular cryo–electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. They pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.
Half a century of amyloids: past, present and future (Chem. Rev.)
Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-beta architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fueled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.
NMR Methods for Structural Characterization of Protein-Protein Complexes (Front. Mol. Biosci.)
Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here Venditti V. et al. discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.
Structure of human GABA(B) receptor in an inactive state (Nature)
The human GABA(B) receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA(B) receptor has two subunits, GABA(B1) and GABA(B2), that are structurally homologous but perform distinct and complementary functions. GABA(B1) recognizes orthosteric ligands, while GABA(B2) couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here O.B. Clarke, J. Frank, Q.R. Fan et al. present a near full-length structure of the GABA(B) receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Their structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. They also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity. The structure of the GABA(B) receptor in an inactive state reveals, amongst other features, a latch between the two subunits that locks the transmembrane domain interface, and the presence of large phospholipids that may modulate receptor function.
Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy (Nat. Commun.)
Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of inter- actions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. M. Baldus et. al. present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed them to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Their results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.
Quote of September
“Extraordinary claims require extraordinary evidence.”Carl Sagan