1 Quo Vadis NMR? CANT MUP-I LTP-1 d(GCGAAGC) CSP-1 δ subunit of RNA polymerase Svatý   Petr   je   v   době   Neronova   pronásledování   křesťanů   přesvědčen   svými   stoupenci,   aby   odešel   z   Říma   do   bezpečí.   Cestou  potkává  Ježíše  Krista,  který  kráčí  do  města,  a  Petr  se  ho   překvapeně   ptá:   „Quo   vadis,   Domine?“   (Kam   kráčíš,   Pane?),   „Když   ty   opouš6š   můj   lid,   jdu   do   Říma,   aby   mě   ukřižovali   podruhé.“,   odpovídá   mu   Ježíš.   Poté   se   Petr   obrací   nazpět   a   později   umírá   na   kříži   stejně   jako   jeho   Pán.   Z  pokory   však,   aby   Ježíše  nenapodoboval,  si  Sv.  Petr  vyprosí,  aby  jej  ukřižovali  jinak  –   hlavou  dolů.   2 NMR E = h.ν ω = γ.Bo E 1945 . . . . 1952 3 ν = γ.Bo /2π.(1 - σ) ~ 10 – 1000 MHz 4 ν = γ.Bo./2π.(1 - σ) Classical Spectroscopy: dν/dt or dB/dt 5 Fourier Transform Joseph Fourier (1768 – 1830) 6 Jean Baptiste Joseph Fourier born Auxerre, March 21, 1768 died, Paris, May 16, 1830 He took a prominent part in his own district in promoting the revolution, and was rewarded by an appointment in 1795 in the Normal school, and subsequently by a chair in the Polytechnic school. Fourier went with Napoleon on his Eastern expedition in 1798, and was made governor of Lower Egypt. After the British victories and the capitulation of the French under General Menou in 1801, Fourier returned to France, and was made prefect of Grenoble, and it was while there that he made his experiments on the propagation of heat. He moved to Paris in 1816. In 1822 he published his Théorie analytique de la chaleur, in which he shows that any functions of a variable, whether continuous or discontinuous, can be expanded in a series of sines of multiples of the variable - a result which is constantly used in modern analysis. J.W.Cooley and J.W.Tukey, Math. Comp. 1965, 19, 297 Fast Fourier Transform 7 1D NMR 2D NMR 3D NMR nD NMR 8 69   Felix  Bloch   (1905-­‐1983)   Edward  M.  Purcell   (1912-­‐1997)   Physical  Review  69,  37  (1946)   Physical  Review  69,  127  (1946)   NMR   9 10 Chemistry Physics Biology Medicine NMR Nuclear Magnetic Resonance 68 years 1945 – 2014 11 Richard R. Ernst (1933 - *) Swiss Federal Institute of Technology (ETH), Zürich, Switzerland Nobel Prize in Chemistry 1991 “for his contributions to the development of the methodology of high resolution nuclear magnetic resonance (NMR) spectroscopy” Kurt Wüthrich (1938 - *) Swiss Federal Institute of Technology (ETH), Zürich, Switzerland and The Scripps Research Institute, La Jolla, USA ”for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution”. Nobel Prize in Chemistry 2002 12 Nobel Prize in Physiology or Medicine 2003 Paul C. Lauterbur (1929 - 2007) Biomedical Magnetic Resonance Laboratory, University of Illinois, Urbana, USA Sir Peter Mansfield (1933 - *) Department of Physics, University of Nottingham, UK for their discoveries concerning "magnetic resonance imaging" Birthday cake Plants Spinal cord µm resolution Human Brain AIDS dementia 13 1945  Detection of NMR signals in bulk materials 1949  Discovery of chemical shift 1950  Discovery of spin-spin coupling 1952  Bloch and Purcell receive Nobel Prize 1953  First commercial NMR spectrometer (Varian 30MHz) 1966  Fourier transform (FT) techniques introduced – R.R. Ernst 1971  Two-dimensional (2D) NMR concept suggested – J. Jeener 1973  Zeugmatography: first two-dimensional NMR image - P. Lauterbur 1974  2D-NMR techniques developed – R.R. Ernst 1985  First 3D structures of proteins from NMR data – K. Wüthrich 1991  Ernst receives Nobel Prize 2002  Wüthrich receives Noble Prize 2003  Lauterbur and Mansfield receive Nobel Prize 2009  1 GHz spectrometer (Bruker) Milestones of NMR History 14 15 1967 90 MHz 1970 270 MHz 1979 400 MHz 1983 500 MHz 1995 800 MHz 1998 700 MHz 1999 750 WB 2000 800 US2 2001 900 MHz Magnet history 2005 950 MHz 2009 1000 MHz 16 NMR as an eminent tool for structural and system biology CANT MUP-I LTP-1 d(GCGAAGC) CSP-1 δ subunit of RNA polymerase 17Features of Rous Sarcoma Virus capsid revealed by cryo electron microscopy and image reconstruction of the virion. EM microscopy X-ray MS spectroscopy A real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome 2.6-MD α6β6 heterododecameric fatty acid synthase from Thermomyces lanuginosus at 3.1Å resolution 18 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 500 1000 1500 2000 #ofproteins # of amino acids Human genome Solution global fold of the monomeric 723-residue (82kDa) enzyme malate synthase G from Escherichia coli (Tugarinov V, Choy WY, Orekhov VY, Kay LE., Proc Natl. Acad. Sci U.S.A., 18, 102:622-7, 2005). 723 aa as of September 16, 2014 X-­‐ray   85540   1559   4551   5   91655   NMR   9291   1093   220   7   10611   ELECTRON   MICROSCOPY   580   67   190   0   837   HYBRID   63   3   2   1   69   other   159   4   6   13   182   Total   95633   2726   4969   26   103354   PDB Current Holdings Breakdown PROTEINS NA PROTEINS/NA complexes OTHERS TOTAL 19 NMR MD simulation 20 NMR of Biomacromolecules 2005 – 82 kDa Vitali Tugarinov, Wing-Yiu Choy, Vladislav Yu. Orekhov, and Lewis E. Kay Solution NMR-derived global fold of a monomeric 82-kDa enzyme, PNAS 2005 102: 622-627; published online before print January 6 2005, 10.1073/pnas.0407792102 Williamson, M.P. Havel, T.F. Wuthrich, K. Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry. J.Mol.Biol. 182. 295-315 ,1985 1985 – 6.2 kDa 21 1H 99.98 % active in NMR 12C 98.9 % non-active in NMR 13C 1.1 % active in NMR 15N 0.3 % active in NMR 31P 100.0 % active in NMR Natural Abundance of NMR isotopes Isotope labeling by molecular biology methods Isotope labeling of RNA In vitro transcription with T7 RNA polymerase using isotopicaly labeled 5'-triphosphates (NTPs) as substrates 22 Production of 13 C,15 N-labeled RNA E. coli fermementation with N-15 amonium sulfate and/or C-13 glucose Isolation of rRNA Nuclease P-1 degradation of rRNA to rNMPs Enzymatic or chemical phosphorylation to rNTPs ATP GTP UTP CTP RNA oligomer synthesis on a DNA template using T7 RNA polymerase23 24 2D HSQC spectra N-terminal domain of M-PMV capsid protein How Do We Go From Spectra to A Structure? Basic principles of 3D structure determination by NMR ? Resonance  Assignments   25 3D TOCSY-HSQC 3D HCC(CO)NH-TOCSY 3D CBCA(CO)NH 3D CBCANH 2D HSQC N Ca Cﾕ N Ca Cﾕ H O H OH CH3 HCH CH3CH3 N H Basic principles of 3D structure determination by NMR 4.1Å 2.9Å NOE CαH NH NH CαHJ NOE - a through space correlation (<5Å) - distance constraint Coupling Constant (J) - through bond correlation - dihedral angle constraint Chemical Shift - very sensitive to local changes in environment - dihedral angle constraint Dipolar coupling constants (D) - bond vector orientation relative to magnetic field - alignment with bicelles or viruses D 26 DAB(θ,φ) = Da(3cos2θ - 1) + 3/2 Dr(sin2θ cos2φ)] Bo Non-averaging of DD interactions in aligned media Ø Dipolar couplings and induced changes of chemical shifts (relative orientation of atom-atom vectors, dynamics) RDC - residual dipolar couplings CSI - induced changes of isotropic chemical shifts 27 TALOS (Cα, Hα, Cβ, C’, N) 3D 15N-NOESY 3D 13C-NOESY inter-proton distance restraints dihedral restraints (ψ, φ) dipolar couplings restraints (DNH, DCαHα) 2D-IPAP 15N-1H HSQC J-mod. 13C-1H CT- HSQC helical hydrogen bonds generation of structures refinement of structures MD MD info on mobile regions Example of NMR structure determination 28 29 ETOTAL = Echem + wexpEexp Eexp = ENOE + Etorsion + EH-bond + Egyr + Erama + ERDC + ECSA + Epara Echem = Ebond + Eangle + Edihedral + Evdw + Eelectr •  Echem : (a priori knowledge ) primary structure, topology, covalent bonds, dihedral angles (harmonic), etc. - non-covalent van-der-Waals forces: Lennard-Jones potential - electrostatic interactions - Coulomb potential etc. •  Eexp : experimental constraint terms Recent potential energy terms used in MD: •  dipolar couplings (Erdc) •  radius of gyration (Egyr) •  CSA (ECSA) •  side chain conformational database torsion angle potentials (Erama) •  paramagnetic relaxation enhancement module (Epara) Potential Energy Terms Mason-­‐Pfizer  Monkey  Virus   M-­‐PMV   q  1970:  Chopra,  H.C.,  Mason,  M.M.  (mammary  carcinoma  of  a  female   rhesus  monkey  -­‐  Macaca  mulana)   q  Retroviridae,  Oncovirinae,  Betaretrovirus   q  D  type    /  C  type  (HIV)     30 31 Assembly of immature viral particles C-type HIV-1 M-PMV D-type 32 N-terminal domain of M-PMV capsid protein β1 α1 β2 α2 α3 α4 α5 α6 Restraint Information Distance restraints 2246 Hydrogen bonding restraints 124 Torsion Angle restraints (φ/ψ) 107/107 Residual Dipolar Couplings 130 Average rms deviation from experimental restraints Distance restraints (Å) 0.023 ± 0.001 dihedral angle restraints (°) 0.552 ± 0.067 residual dipolar couplings (Hz) 1.811 ± 0.061 RDCs Q factor 0.225 ± 0.008 Violations distance violations > 0.5Å 0 dihedral angle violations > 5° 0 Pairwise Cartesian RMS deviation (Å) ordered all heavy atoms 1.60 ± 0.23 ordered backbone heavy atoms 1.11 ± 0.23 ordered all heavy atoms 1.24 ± 0.10 helices only ordered backbone heavy atoms 0.74 ± 0.11 helices only 33 PDB 1GWPPDB 3BP9 PDB 1AK4 PDB 1G03 PDB 1EM9 PDB 2KGF 34 MLV x-ray RSV X-ray HIV nmr HIV x-ray M-PMV nmr HTLV nmr Pro-1 Asp-57 35 MLV CA-NT hexamer by x-ray (Mortuza, G.B. et al. JMB 376, 1493-1508, 2008) M-PMV CA-NT hexamer model from nmr data Fullerene model for the conical capsid, with CA hexamers (Ganser-Pornillos, B.K. et al. COSB, 18, 203-217, 2008) 36 Dynamics and molecular motions Heteronuclear relaxation 15N, 13C T1 – spin-lattice relaxation time– fast motions T2 – spin-spin relaxation time – fast & slow motions 1H-15N NOE – fast and some slow motions large ---- small molecules biomacromolecules relaxation 37 Quo vadis? Molecular motions on ps -ns time scale MUP – mouse urinary protein conformational exchange Increased flexibility single motional mode (overall tumbling) Křížová et al. J. Biomol. NMR 28, 369-384, 2004 Pheromones 38 Start X-ray 5 ns 12 ns 16 ns Macek, P., Novák, P., Křížová, H., Žídek, L., and Sklenář, V.: FEBS Letters, 2006, 580, 682-684: Molecular Dynamics Study of Major Urinary Protein – Pheromone Interactions: A Structural Model for Ligand-Induced Flexibility Increase. C D L3 Macek, P., Novák, P., Žídek, L., and Sklenář, V.: J. Phys. Chem. B 2007, 111, 5731-5739: Backbone Motions of Free and Pheromone-Bound Major Urinary Protein Studied by Molecular Dynamics Simulation. MUP (120F) – PDB: 1YP7 10 MUP complexes PDBs: 1QY0,1QY1, 1QY2, 1YP6, 1ZND, 1ZNE, 1ZNG, 1ZNH, 1ZNK, 1ZNL) X-ray Side chains N35 and R60 39 structure of SAMVts1p with RNA: a shape specific recognition Quo vadis? Molecular complexes of increasing size and complexity Oberstraaa et al. Nat. Struct. Biol. 13, 160-167, 2006 40 Quo vadis? 3D protein structure generation from NMR chemical shift data CHESHIRE (CHEmical SHIft REstraints) Michele Vendruscolo, Oxford CS-ROSETTA Ad Bax, Bethesda 11 proteins in the size range of 46–123 residues, yielded results remarkably close (1.3–1.8 Å backbone atom rmsd; 2.1–2.6 Å rmsd for all atoms) to structures previously determined using conventional x-ray crystallography or NMR methods. 16 proteins with known structures; 9 proteins with unknown structures for which only chemical shift assignments but no structural coordinates were available. 56 to 129 residues full-atom models that have 0.7–1.8 Å backboe atom rmsd to the experimentally determined x-ray or NMR structures. 41 Quo vadis? Characterization of weakly interacting molecular networks INPHARMA method Tubulins are targets for anticancer drugs like Taxol and the "Vinca alkaloid" drugs such as vinblastine and vincristine. The anti-gout agent colchicine binds to tubulin and inhibits microtubule formation, arresting neutrophil motility and decreasing inflammation. The anti-fungal drug Griseofulvin targets mictotubule formation and has applications in cancer treatment. epothilone A (E) baccatin III (B) Sanchez-Pedregal et al. Angew. Chem. 44, 4172-4175, 2005 42 Quo vadis? Kinetic processes - Folding of α-lactalbumin Schanda et al. PNAS 104, 11257- 11262, 2007 43 Quo vadis? Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy Hsu et al. PNAS 104, 16516- 1521, 2007 44 Quo vadis? Structure and function of biomacromolecules in living cells or cell extracts NMR solution structure of TTHA1718 in living E. coli cells. Yutaka Ito et al. Nature 458, 102-106, 2009 In cell In vitro 45 Comparison of the three TAR dynamical conformers (green) and ligand-bound TAR conformations (grey). Sub-conformers along the linear pathway linking conformers 1R2, 2R3 and 3R1 are shown in light green, and the direction of the trajectory is shown with arrows. Qui Zhang et al. Nature, 450, 1263 (2007) Quo Vadis Dynamics from Residual Dipolar Couplings Molecular motions on µs time scale HIV TAR 46 O.F. Lange, N.A. Lakomek et al. Science 320, 1471-1475 (2008) Conformational SelectionInduced Fit Recognition mechanisms 46 ubiquitin structures Quo Vadis Dynamics from Residual Dipolar Couplings Molecular motions on µs time scale 47 Wasmer, C., Lange, A., Van Melckebeke, H., Siemer, A.B., Riek, R., Meier, B.H. Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core Science 319, 1523-1526, 2008 Quo vadis? 3D protein structures by solid-state NMR Loquet, A., Bardiaux, B., Gardiennet, C., Blanchet, C., Baldus, M., Nilges, M., Malliavin, T., Bockmann, A. 3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints. J.Am.Chem.Soc. 130, 3579-3589, 2008. NMR X-ray 54.7°magnetic field MAS: 13C –NMR of 13C, 15N labelled peptide in hydrated DMPC bilayer with and without sample spinning [2-13C]glycerol SH3 [U-13C]glycerol SH3 48 Quo vadis? Biomolecular NMR methodology CRINEPT TROSY SAIL isotope labeling stereo-array isotope labeling cell-free expression Kainosho et al. Nature 440, 52-57, 2006Riek et al. PNAS 96, 4918-4923, 1999Pervushin et al. PNAS 92, 12366-12371, 1997 [u-15N, 75% 2H]-labeled S. aureus aldolase a 110 kDa protein, τc > 70 ns 49 Single-scan nD acquisition" Quo vadis? Biomolecular NMR methodology 50 Quo vadis? Non-linear sampling 2D projection of 3D HNCO Linear sampling 20 hours 2D projection of 3D HNCO Non-linear sampling 3 hours δ subunit of RNA polymerase with an unstructured C-tail (57 aa) 51 Quo vadis? Biomolecular NMR methodology DNP – Dynamic Nuclear Polarization In Situ Temperature-Jump DNP Griffin et al. J. Amer. Chem. Soc. 131, 12-13, 2009 800 ms laser pulse 40 s freezing by N2 gas 52 Quo vadis NMR? Current challenges • study of molecular complexes of increasing size and complexity • characterization of weakly interacting molecular networks • investigation of structural preferences in natively unstructured proteins • observation of kinetic processes and excited state conformations involved in protein folding, binding, and allosteric signal transduction • studies of structure and function in living cells or cell extracts • studies of molecular motions on µs -ms time scale in complex assemblies • structure determination in solid state polycrystalline samples • structure determination of membrane proteins using solid state technology 53 Quo vadis NMR? Forward, future is bright 3D HsCNbCHb experiment MQ-TROSY t3δ δ σ σ 180(C6,C8)180(C1') H C N G σ 2δ 180(C1',C6,C8) t1 180(N9,N1) τ−σ 180(H1') Δ+t2/2 Δ−t2/2 ϕ1 ϕ3 ϕ6 ϕ4 ϕ7 ϕ5 ϕ2 σ τ−σΔ−δΔ−δ cpd cpd time ~ 10 – 100 ms Magic Spin Gymnastics 54 NMR Laboratory 500 MHz 600 MHz 1995 2001 Bruker Avance 300 MHz 1999 55 Structural Biology prof. Vladimír Sklenář Programme coordinator Vision 57 Structural Biology Program will integrate information on the structure of biologically important macromolecules - proteins, nucleic acids and their complexes. The aim is to obtain the knowledge necessary to understand the basic functions and life processes at the molecular and cellular levels. Progressive high-resolution methods of structural analysis such as X-ray diffraction, nuclear magnetic resonance, cryo-electron microscopy and tomography will be used in combination with modern methods of molecular modelling, theoretical chemistry, and bioinformatics. Time variations of three-dimensional structures, supplying essential information indispensable for painting a dynamic picture of key cellular functions, will be investigated in detail. X-ray NMR Cryo-EM & tomography Vision 58 A new integrated infrastructure built within CEITEC will be used to develop modern methods of structural biology and to extract molecular data crucial for biochemical and biomedical applications. The Structural Biology programme is aimed at achieving European competitiveness, stimulate regional development and facilitate biomedical research and biotechnologies. At the application level, its results will facilitate developments of the next-generation diagnostic and therapeutic strategies for the treatment of human diseases and solutions of health problems. integrative approaches Vision 59 NMR X-ray crystallography Cryo-electron microscopy and tomography Biophysical investigations X-ray crystallography Mass spectrometry Structural Bioinformatics Czech Infrastructure for Integrative Structural Biology Goal: National Affiliate Centre (approved 2011) INSTRUCT Centre (2015) INSTRUCT - Integrated Structural Biology Infrastructure for Europe ESFRI project Vision 60 Bioinformatics Computational Chemistry Czech ELIXIR node Czech Republic National Roadmap for Research Infrastructures ELIXIR - Infrastructure for biological information in Europe ESFRI project Research objectives "   Investigation of the role of RNA in development and human diseases. "   To study the therapeutical aspects of recognition and adhesion phenomena in host-pathogen interactions. "   The visualization and modification of biological objects including tissues, cells, cellular structures, and biomolecules. "   The development of new methodologies for investigating the structure, interactions, and dynamics of biomolecules. "   High throughput structural characterization of macromolecular assemblies by single-crystal diffraction. 61 Research objectives 62 "   To establish a bioinformatics laboratory focussing on gene expression profiling and sequence analysis. "   To establish a high-end cryo electron microscopy laboratory for highly sophisticated 3D imaging studies for structural biology at the cellular level. "   Investigations of the structure and interactions of biomacromolecules and their relation to the functions of living systems, disease and therapy. "   Investigations of the behaviour of natural and chemically modified biomacromolecules at electrically charged surfaces linked to the development of novel electrochemical biosensors and bioassays. "   The enhancement of the theoretical background to the design, synthesis and clinical use of novel anticancer and antiviral therapeutics. Work Packages WP-3-1 | Structural and molecular biology of RNA WP-3-2 | Structural and molecular biology of host-pathogen interactions WP-3-3 | Nanobiotechnology for Health WP-3-4 | New methods for structural biology WP-3-5 | Protein crystallography WP-3-6 | Gene expression profiling and sequence analysis WP-3-7 | Cryo electron microscopy and tomography of cellular biopolymers WP-3-8 | Biophysics and biophysical chemistry of biopolymers and their interactions 63 Research Groups | Research Group Leaders "   RG-3-01 | Bioinformatics | Hedi Hegyi "   RG-3-02 | CD Spectroscopy of Nucleic Acids and Proteins | Michaela Vorlíčková "   RG-3-03 | Cryo-EM | Jürgen Plitzko "   RG-3-04 | Glycobiochemistry | Michaela Wimmerová "   RG-3-05 | RNA Quality Control | Štěpánka Vaňáčová "   RG-3-06 | Nanobiotechnology | Petr Skládal "   RG-3-07 | Biomolecular NMR Spectroscopy | Vladimír Sklenář "   RG-3-08 | RNA-based regulation of gene expression | Peter J. Lukavsky "   RG-3-10 | Structural biology of gene regulation | Richard Štefl "   RG-3-12 | Structural Virology | Pavel Plevka "   RG-3-13 | Structure and Dynamics of Nucleic Acids | Jiří Šponer "   RG-3-14 | Structure and Interaction of Biomolecules at Surfaces | Miroslav Fojta "   RG-3-15 | Computational Chemistry | Jaroslav Koča 64 Core Facilities Josef Dadok National NMR Centre 65 •  950 MHz NMR with a cryoprobe for high-resolution spectroscopy in liquids •  850 MHz NMR with a cryoprobe for high-resolution spectroscopy in liquids •  700 MHz NMR with a cryoprobe for high-resolution spectroscopy in liquids •  700 MHz NMR for high resolution NMR in liquids and solids •  Upgrade of existing 600 MHz NMR with a cryoprobe for highresolution spectroscopy in liquids •  Upgrade of existing 500 MHz NMR with a cryoprobe for highresolution spectroscopy in liquids and solids •  Operational since November 2012 Core Facilities X-ray/Bio-SAXS 66 •  Crystal preparation -  Robotized crystallization laboratory -  Crystallization laboratory equipment -  X-ray and optical protein scanner •  2 RIGAKU diffractometers with rotating anode -  One robotized protein diffractometer -  Universal mono-crystal diffractometer Core Facilities Biomolecular Interactions 67 •  Equipment planned for Biomolecular interactions CF –  microcalorimeter AutoITC200 (GE Healthcare) – in full operation since March 2013 –  Semi-automated SPR system (GE Healthcare SPR Biacore T100, T200?) - tender in preparation –  SPR biosensors for multiplex measurements (Horiba Openplex, BioRad ProteOn XPR36? - tender in preparation •  In the meantime, instrumentation purchased from other resources is offered: –  microcalorimeters VP-ITC, VP-DSC, ITC200 (GE Healthcare) –  SPR biosensor Biacore 3000 (GE Healthcare) –  Analytical ultracentrifuge ProteomLam XLI (Beckman-Coulter) –  CD spectrophotometer J-815 (Jasco) equipped with fluorescence and stopped-flow (Biologic) AutoITC200 Core Facilities Nanobio 68 Currently available instrumentation: •  Atomic force microscope –  Nanowizard3 + ForceRobot300 (JPK) –  to be mounted on the confocal fluorescence microscope IX81/FV1000 (Olympus) •  Deposition and immobilization of biomolecules –  sciFLEXARRAYERS S1 and S3 (Scienion) Tenders: •  in progress: fully automated AFM (edu level), electrochemical / nanolithographic modules for current AFM Ntegra Vita •  in preparation: electrochemical analyzer (entry level, repeated) •  high-resolution fluorescence scanner Core Facilities cryo-EM 69 •  Titan Krios (the voltage range of 80 to 300kV) for high throughput single particle acquisition as well as for cellular tomography purposes •  Tecnai F20 for computer assisted single particle acquisition or tomography of thinner objects (small prokaryotic cells or sections) as well as for tomography of macromolecular complexes to determine their higher-order structure. •  FIB Versa 3D for micromachining and milling thin lamellas of cryo specimens, such as intracellular compartments, suitable for cryoelectron tomography •  Vitrobot Mark IV for reproducible and environmentally controlled sample preparation of frozen hydrated EM grids of cells or purified and isolated macromolecular complexes Vitrobot Mark IV