Cryo-Electron Microscopy Pavel Plevka Transmission Electron Microscope microscope transmission_electron_microscope Electron source: Thermal emission from heated cathode Focussing: Electro-magnetic Lenses Detection: Phosphor screen or CCD camera (former times: negative) Vacuum! Pro & Con of cryo-EM •Pro •Short wavelength => high resolution •Strong interaction with materials => good contrast •Electromagnetic lenses => standard optics (in contrast to X-ray crystallography) •High intensity is easy to produce •Inner structure of biomolecules is accessible • •Con •High vacuum requires special treatment of sample •Sample has to be thin to avoid 100% absorption •Electron beam damages biological samples => short measurements => low contrast of biomolecules • • nl = 2d sinq Bragg’s law Rayleigh criterion Why electrons? Spektrum Visible Light: l = 400 – 600 nm Electrons: l = 0.002 – 0.004 nm Sample Preparation - Staining ÞTo increase contrast: heavy atoms interact with electrons stronger than biomolecules (C, N, O, S, P) • •Positive Staining • treat sample with solution of salt like uranyl acetate, lead citrate, osmium tetraoxide – object is black on light background •Negative Staining • place sample on dried film of heavy metal salt – object is light spot on black background •Shadowing • spray thin layer of heavy metal on sample to produce a shadow • •Disadvantage: •Size of stain reduces resolution to about 20-30 Å Alternative: Cryo-EM • to avoid harsh staining which may change the structure of your sample • stabilization of sample by rapid freezing of sample in liquid ethane to form vitreous ice • into electron microscope at low temperatures to keep sample stable in hydrated state in vacuum • thickness of ice layer as small as possible! • raw Advantage: • sample structure is unchanged • inner structures of molecules are accessible Types of Samples •Periodic arrangement • => 2D electron crystallography • small or membrane proteins < 200 kDa • resolution up to 2.5 Å • •Random arrangement • => single particle technique • macromolecular complexes > 200 kDa • up to atomic resolution • •Large Organelles (Golgi, ER), whole cells • => tomography • resolution > 4 Å bR_heptamer_big Fig_5 Ribosome Bacteriorhodopsin For a given sampling frequency, the maximum frequency you can accurately represent without aliasing is the Nyquist frequency, which equals one-half the sampling frequency, as shown by the following equation. Macromolecules in water / vitreous ice are phase objects Signal to noise ratio Classification and averaging (principal component analysis) 3D Reconstruction When the angles between the different classes are known (estimated), a 3D model can be calculated. EM_1 Iterative Process: 3D model is used to generate 2D images which are fed into statistical analysis of images (alignment and classification). And then? Try to interpret 3D map, e.g. try to fit known crystal structures into electron density map Cryo TEM sample preparation using Vitrobot phi812_scheme_1.png phi812_scheme_2.png phi812_scheme_3.png test Infection cycle life_cycle_with_questions_3.jpg life_cycle_with_questions_3_NOQ.jpg Virus replication factory ? … aspiring to answer basic biological questions about picornavirus life cycle. It is well established that picornaviruses enter cells by receptor mediated endocytosis. However, it is not known how is the picornavirus ssRNA genome delivered from virions across the membrane of the endosome into cell cytoplasm. That is our first research question. When in the cytoplasm the genome is translated into a polyprotein that is then cleaved into functional subunits. The viral proteins induce formation of so called virus-replication factories that contain lipid vesicles derived from intra-cellular membranes, viral and cellular proteins, ribosomes and viral RNAs. We will use modern cryo-electron tomography approaches to characterize the factories on molecular level. In addition we will structurally characterize picornavirus genome replication and recombination – processes that happen in the vicinity of the replication factories. Finally, we will determine picornavirus virion assembly mechanism. in situ cryo-electron tomography EM_grid_photo.png cells_pleiomorphic.png A picture containing object, clock Description automatically generated tomo7_v6_legend A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated A picture containing text, old, vintage Description automatically generated Endocytosis of VLDL - native cargo of VLDLR Jeov et al. 2005 Diagram Description automatically generated Wiskostatin inhibits enterovirus infection Genome release intermediates of echovirus 18 Buchta et al. 2019 Open particles of echovirus 18 fig2_1_5col.pdf Buchta et al. 2019 Harutyunyan et al. 2013 Human rhinovirus 2 Picornaviridae Slow bee paralysis virus Iflaviridae Škubník et al. 2021 Kashmir bee virus Dicistroviridae Mukhamedova et al. 2021 Echovirus 30 Picornaviridae Buchta et al. 2019 Acidic pH induces genome reorganization Virions at neutral pH Activated particles at acidic pH Škubník et al. 2021 Deformed wing virus (Iflaviridae) A picture containing colorful Description automatically generated Neutral pH Virion Empty particle Full particle Acidic pH Buchta et al. 2019, Sukeník et al. 2021 1. Ktere z nize uvedenych zareni a castic interaguje nejsilneji s biologickym materialem? a) elektrony b) viditelne svetlo c) paprsky X 2. Jake je v soucasnosti nejvyssi rozliseni dosazene pri studiu makromolekul pomoci kryo-elektronove mikroskopie? a) 1.0 Å b) 0.23 μm c) 0.5 nm 3. Jake je nejvyssi mozne rozliseni obrazku, ktery ma velikost pixelu 1.1Å? a) 1.1 Å b) 2.2 Å c) 3.3 Å