COVID-19 Measures
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.
Core Facilities
CEITEC Core Facilities
News
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Upgrade of the workflow for cryo-electron tomography and microscopy
The CIISB Cryo-electron microscopy core facility at CEITEC Masaryk University is expanding its services in the sample preparation for electron microscopy. The facility has recently acquired high-pressure freezer Leica EM ICE for vitrification of bulky biological specimen (up to 200 mm thickness). In addition, the freeze-substitution unit Leica EM AFS2 for resin embedding of the high-pressure frozen samples and the ultramicrotom Leica EM UC7 with the adapter for cryo-ultramicrotomy were purchased in order to provide the facility users with the complete workflow for preparation of thin section samples for both room-temperature electron microscopy and cryo-electron microscopy.
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Emerging Topics in Biomolecular Magnetic Resonance – Nick Cox & Kendra Frederick
The series Emerging topics in Biomolecular Magnetic Resonance will continue on July 9th at 16:00 CEST with the following lecturers and topics:
Nick Cox (Australian National University): Spin state evolution during the biological water splitting reaction
Kendra Frederick (UT Southwestern): In cell structural biology enabled by DNP MAS NMR
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Emerging Topics in Biomolecular Magnetic Resonance – 4th edition
The series Emerging topics in Biomolecular Magnetic Resonance will continue on June 25th at 16:00 CEST with the following lecturers and topics:
Melinda J. Duer (University of Cambridge): Understanding extracellular matrix disease states with solid-state NMR?
Claudio Luchinat (University of Florence): NMR for metabolomics: again the ‘second best’ technique?
Highlights of Coronavirus Structural Studies
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Structure of the full SARS-CoV-2 RNA genome in infected cells
SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs or vaccines with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on predicted RNA structures in SARS-CoV-2 and most putative regulatory sequences are uncharacterized. Here we report the secondary structure of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq). Silvi Rouskin et al. reveal previously undescribed structures within critical regulatory elements such as the genomic transcription-regulating sequences (TRSs). Contrary to previous studies, their in-cell data show that the structure of the frameshift element, which is a major drug target, is drastically different from prevailing in vitro models. The genomic structure detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.
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Map of SARS-CoV-2 spike epitopes not shielded by glycans
The severity of the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, calls for the urgent development of a vaccine. The primary immunological target is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface to mediate viral entry into the host cell. To identify possible antibody binding sites not shielded by glycans, Gerhard Hammer et al. performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation and generic antibody binding signatures, they recover known epitopes on S and reveal promising epitope candidates for vaccine development. They find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics in epitope mapping.
Research Highlights
the best of science obtained using CIISB Core Facilities
Nature Communications 2020
Structure of the RcGTA particle and organization of segments of the R. capsulatus genome encoding protein components of RcGTA particles. a Cryo-EM reconstruction of a native particle of RcGTA from R. capsulatus strain DE442 calculated from 42,242 particle images. The left part of the panel shows the complete particle, whereas on the right the front half of the particle has been removed to show DNA and internal proteins. Individual proteins in the density map are colored according to the gene map in panel b. Yellow mesh highlights the structural organization of capsid proteins within the RcGTA head. The inset shows an example of a two-dimensional class average and an electron micrograph of an RcGTA particle. The scale bar within the inset represents 20 nm. b Gene map of three genome segments encoding fourteen structural proteins of RcGTA particles. c Cryo-EM reconstruction of an RcGTA particle from R. capsulatus strain DE442 with T = 3 quasi-icosahedral head. The reconstruction is based on 1076 particle images. The structure is at the scale of those shown in panel a. The inset shows an example of a two-dimensional class average and an electron micrograph of RcGTA particle with an icosahedral head. Scale bar represents 20 nm. d Organization of capsomers in the oblate capsid of RcGTA. Capsomers forming one fifth of the capsid are highlighted in different colors and marked with P for pentamer and H for hexamer.
Significance
Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.
Bárdy,P., Füzik, T., Hrebík, D., Pantůček, R., Beatty, T.J. and Plevka, P: Structure and mechanism of DNA delivery of a gene transfer agent, Nat. Commun. (2020) 11, 3034.doi.org/10.1038/s41467-020-16669-9
Int. J. Biol. Macromol. 2020
Crystal structure of BP39L and CV39L. (A) Crystal structure of BP39L shown in secondary structure representation (β-strands in yellow and loops in green) (B) Surface representation (yellow) of BP39L color-coded according to putative binding sites (blue). (C) Crystal structure of CV39L shown in secondary structure representation (β-strands in violet, α-helices in cyan and loops in pink). (D) Surface representation (violet) of CV39L color-coded according to putative binding sites (blue), α-helices shown in cyan.
Michaela Wimmerova Research Group
Significance
Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria – BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed β-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.
Sykorova, P., Novotna, J. Demo, G., Pompidor, G., Dubska, E., Komarek, J., Fujdiarova, E., Houser, J., Haronikova, L., Varrot, A., Shilova, N., Imberty, A., Bovin, N., Pokorna, M., and Wimmerova, M.: Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed beta-propeller fold, Int. J. Biol. Macromol. 2019, 152, 1113-1124, doi.org/10.1016/j.ijbiomac.2019.10.200
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.
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First Experiments in Structural Biology at the European X-ray Free-Electron Laser
Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.
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In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer
Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharidevan. Kugelen et. al. report an electron cryo-microscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, they deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, they present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryo-tomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
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Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS
Technical developments are unifying molecular and cellular biology. A recent electron cryo-tomography study by von Kugelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.
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Mapping Structural Dynamics of Proteins with Femtosecond Stimulated Raman Spectroscopy
The structure–function relationships of biomolecules have captured the interest and imagination of the scientific community and general public since the field of structural biology emerged to enable the molecular understanding of life processes. Proteins that play numerous functional roles in cellular processes have remained in the forefront of research, inspiring new characterization techniques. In this review in Annual Review of Physical Chemistry, Chong Fang and Longteng Tang present key theoretical concepts and recent experimental strategies using femtosecond stimulated Raman spectroscopy (FSRS) to map the structural dynamics of proteins, highlighting the flexible chromophores on ultrafast timescales. In particular, wavelength-tunable FSRS exploits dynamic resonance conditions to track transient-species-dependent vibrational motions, enabling rational design to alter functions. Various ways of capturing excited-state chromophore structural snapshots in the time and/or frequency domains are discussed. Continuous development of experimental methodologies, synergistic correlation with theoretical modeling, and the expansion to other nonequilibrium, photo-switchable, and controllable protein systems will greatly advance the chemical, physical, and biological sciences.
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The regulation and functions of DNA and RNA G-quadruplexes
DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In the paper published in Nature Reviews Molecular Cell Biology, Balasubramanian, S. et al. first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. They then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, they consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, they discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.
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The architecture of the Gram-positive bacterial cell wall
The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor. In Gram-positive bacteria, peptidoglycan is tens of nanometers thick, generally portrayed as a homogeneous structure that provides mechanical strength. S. J. Foster & J. K. Hobbs et.al. applied atomic force microscopy to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The paper published in Nature shows that the mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface, providing information complementary to traditional structural biology approaches.
Quote of July
“Physics is like sex: sure, it may give some practical results, but that's not why we do it.”
Richard P. Feynman