CIISB Newsletter June 2018
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Editorial
Dear Colleagues,
welcome to the second edition of the CIISB Newsletter. This 2018 Spring issue summarises the most important developments at CIISB from December 2017 till May 2018.
CIISB went successfully through the evaluation of European Science Foundation and was included into the European Infrastructure Databank MERIL.
The instrumentation of CIISB is now being gradually upgraded using the funds of the OP VVV project CIISB4HEALTH. The flag ship instrument for cryo-electron microscopy and tomography Titan Krios has been equipped with the latest direct electron detectors Gatan K2 and Falcon III, and Volta phase plate has been installed to improve contrast and quality of the obtained images. The Proteomic Core Facility purchased a new tribrid mass spectrometer and Structural Mass Spectrometry Core facility at BIOCEV acquired a new MALDI-TOF mass spectrometer. In addition, both differential scanning fluorimeters at CEITEC and BIOCEV have been upgraded to enhance their capabilities.
Out of many meetings, I would like to highlight the 2ndCIISB User Meeting, which was organized as a part of the XVth Discussions in Structural Molecular Biology in Nové Hrady, March 22-24, 2018. This traditional conference gathered more than 130 attendees. The recent achievements and progress in cryo-electron microscopy and tomography were discussed during the 1stCEITEC Symposium on recent Advance in Cryo-Microscopy organized in Brno, on April 18, 2018.
It is very rewarding to see an increasing number of papers published with the support of CIISB in journals with high impact factors. This spring, two papers facilitated by Josef Dadok National NMR Centre and Cryo-EM Core Facility were published in highly prestigious periodical Nature Communications by groups of Konstantinos Tripsianes and Pavel Plevka. Their results are described in Research Highlights. Results of other important publications can be found in the Research Highlight Section on the CIISB web.
I wish you enjoyable summer.
Vladimír Sklenář
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CIISB was included into the MERIL database
Research infrastructure CIISB went successfully through the evaluation of European Science Foundation (link http://www.esf.org/) and was included between European research infrastructures into the database MERIL (Mapping of the European Research Infrastructure Landscape). We have joined other research infrastructures that operate on the basis of transparent „open access“ approach. In general, the database serves as a background for ESFRI in the mapping of EU research infrastructures and also it is a confirmation that the research infrastructure meets European criteria. Moreover, it serves as another tool where to present services to potential users and to establish co-operation. More about MERIL here (link https://portal.meril.eu/meril/).
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New Orbitrap Fusion Lumos Tribrid Mass Spectrometer installed at CEITEC
New Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Scientific) has been installed at the Proteomics Core Facility, CEITEC MU. The new instrument equipped by three mass analyzers (a quadrupole, linear ion trap, an Orbitrap) and four fragmentation techniques (CID, HCD, ETD, EThcD) significantly improves our capabilities and flexibility in data dependent and data independent acquisition modes. New technologies (such as Advanced Peak Determination or Synchronous Precursor Selection) bring large increase of the number of peptides and proteins identified and improve accuracy in quantitative studies using isobaric mass tags (TMT).The purchase of the instrument was financed from the European Regional Development Fund-Project „CIISB4HEALTH“ (No. CZ.02.1.01/0.0/0.0/16_013/0001776).
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Transmission electron microscope Titan Krios
Transmission electron microscope Titan Krios at Cryo-electron microscopy and tomography core facility CEITEC MU has been recently upgraded with phase plate, and two last generation direct electron detectors (FEI Falcon3EC, Gatan K2). Phase plate significantly increases contrast in cryo-electron microscopy data and allows to collect data at in-focus conditions. Moreover, increased contrast extends applicability of cryo-electron microscopy to high-resolution structural characterization of proteins and protein complexes which are around 100kDa in size. Both newly acquired direct electron detectors operate in electron counting mode, which provides superior image quality. Besides, the Gatan K2 detector position is behind the post-column energy filter and allows zero-loss imaging. Such application is primarily beneficial for imaging of thicker samples such as vitrified cells or focused ion beam micromachined cellular lamellas using cryo-electron tomography for the purpose of structural characterization of biomacromolecular complexes in situ. All upgrades were financed from the European Regional Development Fund-Project „CIISB4HEALTH“ (No. CZ.02.1.01/0.0/0.0/16_013/0001776).
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Upgrades of the differential scanning fluorimetry instruments Prometheus NT.48 at BIOCEV and CEITEC
Recently, both instruments Prometheus NT.48 at BIOCEV and CEITEC, permitting precise measurements of the intrinsic fluorescence of proteins, thereby characterizing their thermal unfolding, have been upgraded to include back-reflection optics, which provides insight into the aggregation processes. The basis for such detection is the aggregation-induced light scattering by particles resulting in loss of light. The combination of thermal stability detection and determination of aggregation onset temperatures allows for the most rapid, precise, and information rich analysis in formulation screenings or protein engineering projects.
The upgraded instruments now can provide comprehensive results for any type of proteins, and are especially suitable for the prediction of proteins stability, for the screening of buffer influence, and for the testing of storage conditions. Part of the upgrade was also increase in the maximum possible temperature from original 95°C to 110°C . This enhancement allows to characterize proteins with higher melting temperature. Both upgrades were financed from the European Regional Development Fund-Project „CIISB4HEALTH“ (No. CZ.02.1.01/0.0/0.0/16_013/0001776).
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Structural Mass Spectrometry CF at BIOCEV acquired a new MALDI-TOF mass spectrometer
The structural mass spectrometry core facility at BIOCEV has expanded its equipment offer (and associated services) with several items.
The core facility now provides access to a new MALDI-TOF mass spectrometer, an Autoflex Speed device from Bruker Corporation. This instrument is suited mostly for the routine detection of peptides and intact proteins. The availability of this new device increases the throughput of analyzed samples, which was previously limited by the lengthy reservation times for accessing the 15T SolariX FT-ICR instrument.
Further, the new excimer laser for fast photo oxidative labeling of proteins will be used for monitoring of fast structural changes of proteins or protein-protein and protein-DNA complexes. A new UHLPC system will be installed in few days to increase the performance of analytes separation. The acquisition of new components was financed from European Regional Development Fund-Project „CIISB4HEALTH“ (No. CZ.02.1.01/0.0/0.0/16_013/0001776).
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1st CEITEC Symposium on Recent Advances in Cryo-Electron Microscopy
Cryo-electron Microscopy and Tomography CEITEC Masaryk University has organized a one day symposium on recent developments in the field of cryo-electron microscopy on 18thApril 2018. The symposium featured eight excellent scientific presentations divided into two sections focused on utilization of cryo-electron tomography and single particle analysis for structural and cellular biology. Prof. Juergen Plitzko from Max-Planck Institute for Biochemistry in Martinsried delivered keynote lecture on the latest technological developments in preparation of thin cellular lamellas from vitrified cells for using focused ion beam micromachining within cryo-electron tomography session. Further, correlative approaches which include cryo-electron tomography have been discussed within this session by other speakers and lecture on approach to achieve atomic resolution with cryo-electron tomography has been delivered by Dr. Florian Schur(IST Austria). Prof. Stefan Raunser (Max-Planck Institute of Molecular Physiology, Dortmund) has presented structures bacterial toxin complexes that permeate the membrane and translocate actin-attacking enzymes into the target cell with session focussing on single particle data analysis approaches. Apart from the lectures focusing of high-resolution virus structures and data analysis, methodological presentation on utilization of Volta phase plate for single particle analysis of small proteins and protein complexes was delivered by Maryam Khoshouei (MPI Martinsried).
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XV. Discussions in Structural Molecular Biology and the 2nd User Meeting of the Czech Infrastructure for Integrative Structural Biology
In March, 2018, the Czech and Slovak community of structural biologists met at the "XV. Discussions in Structural Molecular Biology". This year, the traditional conference was coupled with the 2nd Users Meeting of the Czech Infrastructure for Integrative Structural Biology (http://www.structbio.org/program-xv-discussions). The conference took place at the venue of the Academic conference centre in Nové Hrady, South Bohemia between Thursday afternoon and Saturday lunch on March 22 - 24 2018.
The program has been very intensive. About 130 participants listened to over thirty talks delivered mostly by young investigators or students and during the poster sessions, 60 posters were displayed and discussed. Two sessions were specifically devoted to the 2nd User meeting of the Czech Infrastructure for Integrative Biology, CIISB, and one to General Assembly of the Czech Society for Structural Biology.
Abstracts of all presentations can be found at http://www.xray.cz/setkani/abst2018/abstracts.htm.
At the end of the conference, three prizes were awarded for the best student posters, and three for the best student talks. The next XVI. Discussions are planned for mid-March 2019 again in Nové Hrady. For details see the web page structbio.org or contact the main organizer Jan Dohnálek at dohnalek007@gmail.com.
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An Instruct/CIISB course took place at the CMS, IBT, Biocev on April 5 and 6, 2018
This course/workshop allowed participants, with some prior knowledge and experience in macromolecular crystallography, to access the biophysical measurements section and the crystal handling/X-ray diffraction section of the Instruct CZ / CIISB CMS core facility for three practical work sessions.
The course started with a session of seven lectures (preceded by an introduction by Dr Jan Dohnalek, who introduced the Centre of Molecular Structure, the CIISB and Eric-Instruct). The lectures were aimed at providing a comprehensive introduction to the field of fragment and small molecule screening. Of particular interest were the lectures given by three of the invited foreign speakers, who have contributed extensively to the field.
The workshop part of the course (three half days) allowed the participants to acquire practical experience on how to apply fragment-screening techniques to a real case, using endothiapepsin (generous gift of Prof. A. Heine, U. of Marburg). From published work, three fragments were selected for the practical sessions. In addition, an additional three fragments (never studied before) were used during the dry runs for the preparation of the practical sessions. For pre-screening using biophysical methods, the practical work involved both labelled and label-free microscale thermophoresis, and differential scanning fluorimetry. The practical sessions dealing with crystal delivery to a library of fragments allowed crystal handling and transfer using both Intelliplates as well as the Jena Bioscience plates containing a 96-fragment library. The final session (X-ray diffraction) involved data collection using soaked crystals, in-situ data collection as well as the viewing of (difference) Fourier maps showing fragment density.
In all, the course welcomed 19 registered participants working in 11 countries, with invited speakers from Germany and the U.K., together with the local speakers and course tutors. The session of lectures was well attended, with ca. 30 attendees having signed the attendance sheet (the entire audience, including the registered participants and teachers / tutors is estimated at ca. 70).
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Interaction of TBEV virions with Fab fragments of neutralizing antibody 19/1786. a Cryo-EM micrograph of TBEV virions incubated with Fab fragments of 19/1786. Scale bar represents 100 nm. b Electron-density map of Fab-covered TBEV virion. c Molecular surface of TBEV virion covered with Fab 19/1786 fragments low-pass filtered to 7 Å resolution. E-proteins are shown in red, green, and blue. Fab fragments are shown in magenta (heavy chain) and pink (light chain). Scale bars in band c represent 10 nm. d Footprints of Fab 19/1786 on TBEV surface. e The Fab 19/1786 binds to the domain III at an angle of 135° relative to the axis of the E-protein ectodomain.
Pavel Plevka Research group
Significance
Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, Pavel Plevka and his coworkers present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion.
Fuzik, T. et al. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody. Nature Communications 9, 11, doi:10.1038/s41467-018-02882-0 (2018). doi:10.1038/s41467-018-02882-0
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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
Significance
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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Mon 3 – Fri 7 Sep 2018
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Thu 15 – Fri 16 Nov 2018
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Dzatko, S. et al. Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells. Angewandte Chemie-International Edition 57, 2165-2169, doi:10.1002/anie.201712284 (2018).
Stafkova, J. et al. Dynamic secretome of Trichomonas vaginalis: Case study of beta-amylases. Molecular & Cellular Proteomics 17, 304-320, doi:10.1074/mcp.RA117.000434 (2018).
Haniewicz, P. et al. Molecular Mechanisms of Photoadaptation of Photosystem I Supercomplex from an Evolutionary Cyanobacterial/Algal Intermediate. Plant Physiology 176, 1433-1451, doi:10.1104/pp.17.01022 (2018).
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