EMBO Rep. 2017
Proc. Natl. Acad. Sci. U. S. A. 2017

NMR structure shows how the CTD-interacting domain of Rtt103p recognizes threonine-4 phosphorylated CTD of RNA polymerase II (RNAPII).

Richard Štefl Research group


Phosphorylation patterns of the C‐terminal domain (CTD) of largest subunit of RNApolymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. The solution structure of the Rtt103p CTD‐interacting domain (CID) bound to Thr4 phosphorylated CTD has been obtained by NMR. The structure reveals a direct recognition of the phospho‐Thr4 mark by Rtt103p CID and shows extensive interactions involving residues from three repeats of the CTD heptad. The structural data suggests that the recruitment of a CID‐containing CTD‐binding factor may be coded by more than one letter of the CTD code.

Jasnovidova, O., Krejcikova, M., Kubicek, K. & Stefl, R. Structural insight into recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by Rtt103p. Embo Reports 18, 906-913, doi:10.15252/embr.201643723 (2017).

The virus structure of deformed wing virus of honeybees determined by cryo-electron microscopy. (A) Surface of the virus is rainbow-colored according to its distance from the particle center. (B) Cartoon representation of structure of the P-domain that decorates deformed wing virus surface is rainbow-colored from residue 260 in blue to 416 in red. The background shows image of the deformed wing virus particles from electron microscope.

Pavel Plevka Research group


Honey bee populations in Europe and North America have been decreasing since the 1950s. Deformed wing virus (DWV), which is undergoing a worldwide epidemic, causes the deaths of individual honey bees and collapse of whole colonies. Three-dimensional structures of DWV determined at different conditions shows that the virus surface is decorated with protruding globular extensions of capsid proteins. The protruding domains contain a putative catalytic site that is probably required for the entry of the virus into the host cell. In addition, parts of the DWV RNA genome interact with the inside of the virus capsid. Identifying the RNA binding and catalytic sites within the DWV virion offers prospects for the development of antiviral treatments.

Skubnik, K. ; Novacek, J.; Fuezik, T.; Pridal, A.; Paxton, R. J. ; Plevka, P., Structure of deformed wing virus, a major honey bee pathogen, PNAS, 114, 3210-3215 (2017) DOI: 10.1073/pnas.1615695114

Nucleic Acids Res. 2017
Proc. Natl. Acad. Sci. U. S. A. 2017

Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions

Daniel N. Wilson Research Group


Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

Graf,  M., Arenz, S., Huter, P., Dönhöfer, A., Nováček, J., and Wilson D.N.: Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions. Nucleic Acids Research 45, 2887-2896, doi:10.1093/nar/gkw1272 (2017).

A model of RNA polymerase II bound to the transcription termination factor Rtt103. The structural model was created using integrative structural biology.

Richard Štefl Research group


RNA polymerase II (RNAPII) not only transcribes protein coding genes and many noncoding RNA, but also coordinates transcription and RNA processing. This coordination is mediated by a long C-terminal domain (CTD) of the largest RNAPII subunit, which serves as a binding platform for many RNA/protein-binding factors involved in transcription regulation. In this work, we used a hybrid approach to visualize the architecture of the full-length CTD in complex with the transcription termination factor Rtt103. Specifically, we first solved the structures of the isolated subcomplexes at high resolution and then arranged them into the overall envelopes determined at low resolution by small-angle X-ray scattering. The reconstructed overall architecture of the Rtt103–CTD complex reveals how Rtt103 decorates the CTD platform.


Jasnovidova, O.; Klumpler, T.; Kubicek, K.; Kalynych, S.; Plevka, P.; Stefl, R. PNAS,Structure and dynamics of the RNAPII CTDsome with Rtt103, PNAS 2017 114 (42) 11133-11138; doi:10.1073/pnas.1712450114

J. Am. Chem. Soc. 2017
J. Biol. Chem. 2017

High-resolution structure of a stable G-hairpin calculated from NMR data (PDB ID: 5M1W). (A) Ten lowest-energy structures. Loop residues are colored orange and O4′ atoms are colored red. (B) Schematic representation of hairpin folding topology. Chain reversal arrangement of the backbone and the 3′-to-5′ stacking of the terminal residues are indicated by dark green and magenta arrows, respectively. Anti and syn guanines that form G:G base pairs are colored dark and light blue, respectively.

Lukáš Trantírek Research Group


The first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence is reported. An 11-nt long G-rich DNA oligonucleotide, 5′-d(GTGTGGGTGTG)-3′, corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae adopts a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pairing arrangements. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed. 

Gajarsky, M.;  Zivkovic, M. L. ;  Stadlbauer, P. ; Pagano, B.;  Fiala, R.;  Amato, J.;  Tomaska, L.;  Sponer, J.;  Plavec, J.;  Trantirek, L. Structure of a Stable G-Hairpin JACS 139, 3591-3594, doi:10.1021/jacs.6b10786 (2017)

Structural changes in crystal structure of of AfGcHK sensor protein induced by sodium dithionite soaking (PDB ID 5OHF) and conformational changes revealed by HDX-MS. (A) Two protein chains of the heme domain of the sensor protein observed in crystal structure including alternative B (yellow) occurring with dithionite soaking. (B) Conformational changes revealed by HDX-MS after 60 min of deuteration of the full-length AfGcHK proteins visualized on the protein structure. Differences between the Fe(III)-OH- form (active) and inactive Fe(II) form are color coded: grey - no difference, red - higher and blue - lower levels of deuteration.

Heme-Containing Sensor Proteins Group of Markéta Martínková

Laboratory of Structure and function of biomolecules, Jan Dohnálek

Laboratory of Structural Biology and Cell Signaling, Petr Man 


The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His-183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. The crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms were determined, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry (HDX–MS) the intramolecular signal transduction mechanisms was investigated in full length AfGcHK.  The results suggest that structural changes at the heme proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. For the first time, the conformational changes associated with signal transduction were studied in a full-length globin-coupled oxygen sensor protein and linked to directly observed structural changes in the globin domain. 

Stranava, M.; Man, P; Skálová, T.; Kolenko, P; Blaha, J.; Fojtikova, V .; Martínek, V.; Dohnálek, J.; Lengalova, A.; Rosůlek, M.; Shimizu, T.; Martínková, M.: Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction. J. Biol. Chem. first Published on November 1, 2017, doi: 10.1074/jbc.M117.817023jbc.M117.817023.

Mol. Cell 2017

Cryo-EM Structures of Polyproline-Stalled Ribosomes in the Presence of EF-P (A-C) Schematic representation (A) and cryo-EM reconstructions (B and C) of PPP-stalled ribosome complexes with (B) or without (C) of EF-P (salmon) bound in the E site. (D and E) Cryo-EM density (mesh) of the CCA end of the P-site tRNA (green) from cryo-EM maps in (C) without EF-P (D) and in (B) with EF-P (E), respectively, with aligned fMet (cyan, PDB: 1VY4) (Polikanov et al., 2014).

Daniel N. Wilson Research Group


Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Huter, P., Arenz, S., Bock, L.V., Frister, J.O., Heuer, A., Peil, L., Starosta, A.L., Peske, F., Nováček, J., Berninghausen, O., Grubmüller, H., Tenson, T., Beckmann, R., Rodina, M.V., Vaiana, A.C., and Wilson D.N.: Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P, Moll Cell 68, No. 3., 515-527.e6 DOI: dx.doi.org/10.1016/j.molcel.2017.10.014

Schematic of an intramolecular A) i-motif DNA structure and B) C.C+ base pair. C) Double-staining (PI/FAM) FCM analysis of transfected HeLa cells with the (FAM)-DAP construct (upper left corner). Percentages of viable DNA non-transfected cells, viable DNA-containing cells, non-transfected dead/compromised cells, and transfected dead/compromised cells with DNA are indicated in left-bottom, right-bottom, left-top, and right-top quadrants, respectively. Confocal microscope images of cells transfected with (FAM)-DAP (upper right corner). The green color indicates the localization of (FAM)-DAP. The blue color corresponds to a cell nucleus stained by Hoechst 33342. Imino region of 1D 1H NMR spectra of DAP in vitro in T-buffer (140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl2, pH 7.0) (black) and in-cell (red). Imino region of 1D 1H NMR spectrum of extracellular fluid taken from the in-cell NMR samples after completion of the spectra acquisition (gray). The (in-cell) NMR spectra were acquired at 20oC.

Lukáš Trantírek Research Group


C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Using state-of- the-art in-cell NMR spectroscopy, Lukáš Trantírek and his colleagues from CEITEC Masaryk University in Brno has evaluated the stabilities of i-motif structures in the complex cellular environment. They showed that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. The obtained data show that i-motif stabilities in vivo are generally distinct from those in vitro. Results are the first to interlink the stability of DNA i-motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.

Dzatko, S., Krafcikova, M., Hänsel-Hertsch, R., Fessl, T., Fiala, R., Loja, T., Krafcik, D., Mergny, J-L., Foldynova-Trantirkova, S., and Trantirek, L.: Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells, Angew. Chem. Int. Edit. 2018, in press, DOI: 10.1002/anie.201712284



Automated structure determination using 4D-CHAINS/autoNOE-Rosetta. (a) Logo of 4D-CHAINS algorithm depicting its powerfulness. Chains squeeze the NMR spectrometer to unleash high-quality structures by using a minimal set of 4D spectra and fully automated data analysis. (b) 4D-CHAINS utilizes two complementary experimental datasets, a 4D-TOCSY and a 4D-NOESY, to yield correct assignments for at least 95% of residues and an error rate of less than 1.5% (middle bar; TOCSY-NOESY). (c-d) Performance of different 4D-CHAINS assignment scenarios for a 20 kDa protein structure, α-lytic protease, calculated using autoNOE-Rosetta. (c) Goodness of structure ensembles is measured using the Rosetta all-atom energy function, backbone heavy atom RMSD to X-ray structure and degree of structural convergence. (d) Lowest-energy structures in each ensemble colored as the points in (c) superimposed on the X-ray reference structure (gray).

Konstantinos Tripsianes Resesarch Group


The automation of NMR structure determination remains a significant bottleneck towards increasing the throughput and accessibility of NMR as a structural biology tool to study proteins. The chief barrier currently is that obtaining NMR assignments at sufficient levels of completeness to accurately define the structures by conventional methods requires a significant amount of spectrometer time (several weeks), and effort by a trained expert (up to several months). Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the NMR structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.

4D-CHAINS software is free for non-commercial usage and can be downloaded from https://github.com/tevang/4D-CHAINS

Evangelidis, T. et al. Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra. Nature Communications 9, 13, doi:10.1038/s41467-017-02592-z (2018).

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