03/Nov/2021 Karel Kubíček 1 Cryo EM: From organelles to atomic-resolution structures of molecules inside the cell Credit to Jiří Nováček 03/Nov/2021 Karel Kubíček 2 Diagram Description automatically generated Optical microscope Electron microscope 03/Nov/2021 Karel Kubíček 3 Diagram Description automatically generated Optical microscope Electron microscope 03/Nov/2021 Karel Kubíček 4 Diagram, engineering drawing Description automatically generated FEG - Field Emission Gun Diagram Description automatically generated SEM – skenovací EM, TEM – transmisní/prozařovací EM 03/Nov/2021 Karel Kubíček 6 Diagram Description automatically generated Focused Ion Beam (FIB) + SEM Graphical user interface, text, application, email Description automatically generated 03/Nov/2021 Karel Kubíček 8 A picture containing graphical user interface Description automatically generated TEM – Sample preparation 03/Nov/2021 Karel Kubíček 9 A picture containing text Description automatically generated e.g. uranyl acetate Diagram Description automatically generated Sample vitrification 03/Nov/2021 10 03/Nov/2021 Karel Kubíček 11 A picture containing key Description automatically generated ThermoFisherScientific Grid 03/Nov/2021 Karel Kubíček 12 Text Description automatically generated A picture containing indoor, person, hand, disk brake Description automatically generated A picture containing indoor Description automatically generated 03/Nov/2021 Karel Kubíček 13 A picture containing text, electronics Description automatically generated cryoem101.org Autogrid: grid + C-shape spring (C-clip) + clip ring Auto-loader: up to 12 autogrids 03/Nov/2021 Karel Kubíček 14 A picture containing indoor, tableware Description automatically generated ThermoFisherScientific; MiTeGen 03/Nov/2021 Karel Kubíček 15 A picture containing text Description automatically generated A picture containing text Description automatically generated Note the ice thickness Grids get damaged 03/Nov/2021 Karel Kubíček 16 A picture containing text Description automatically generated A picture containing text Description automatically generated single particles Diagram Description automatically generated Structure determination of biomolecules using TEM Diagram Description automatically generated Structure determination of biomolecules using TEM Diagram Description automatically generated Structure determination of biomolecules using TEM Diagram Description automatically generated Structure determination of biomolecules using TEM Diagram Description automatically generated crYOLO - an application for fast and accurate cryo-EM particle picking You Only Look Once Diagram Description automatically generated Structure determination of biomolecules using TEM 03/Nov/2021 Karel Kubíček 23 https://doi.org/10.1016/j.cell.2019.04.006 Diagram Description automatically generated A picture containing several Description automatically generated Structure determination using single particle cryoEM 1) Sample preparation – 2) vitrification – 3) measurement – 4) particle picking – 5) 2D classification – 6) 3D model reconstruction 03/Nov/2021 Karel Kubíček 24 Micrographs processing 1)Denoising i.Just Another Noise 2 Noise Implementation (JANNI) ii.TOPAZ iii. iii. iii. 2)Lowpass filtering Timeline Description automatically generated 03/Nov/2021 Karel Kubíček 25 Background pattern Description automatically generated with low confidence Original data* After denoising *each micrograph is average of a movie of ~40..60 frames, motion corrected and dose-weighted 03/Nov/2021 Karel Kubíček 26 Particle picking 1.Initial Manual Picking (cyYOLO, Relion, Topaz, cryoSPARC …) 2. i.Selected micrographs (10-100) ii.Fully picked micrographs (80-100 % of particles present) iii. 2.Model Training 3. 3.Full Dataset Picking (X00.000 – X.000.000 particles) A picture containing text Description automatically generated 03/Nov/2021 Karel Kubíček 27 Particle Extraction i)Creates separate image for each individual picked particles ii) ii)Large particles can be reduced by so called binning => speeds up initial calculations iii) Once dataset is “cleaned” and only ”good” particles selected, calculations are repeated using full-scale particles i) i) 03/Nov/2021 Karel Kubíček 28 2D Classification i).alignment (a translation and an in-plane rotation) to map one image onto another ii) ii)images have high noise relative to the signal of particles iii) iii)aligning several similar images to each other then averaging them => image with higher signal to noise ratio iv) iv)the noise is mostly randomly distributed and the underlying image features constant v) v)averaging the intensity of each pixel over several images only the constant features are reinforced. 03/Nov/2021 Karel Kubíček 29 A picture containing white, cabinet Description automatically generated 2.995.892 particles picked from 13.807 micrographs using cryoSPARC template picker 718.639 particles selected after 2D classification 03/Nov/2021 Karel Kubíček 30 3D Reconstruction i)Initial model ii) ii)3D model reconstruction iii) => only a 3D electron density map will be provided 3D structure reconstruction is another (tedious) work: 1.rigid body docking 2. 2.manual model building (semi-automatic) – Coot, Isolde … 03/Nov/2021 31 Chart, line chart Description automatically generated 03/Nov/2021 32 A picture containing flower, plant Description automatically generated Chart, line chart Description automatically generated 03/Nov/2021 33 A picture containing decorated, coelenterate Description automatically generated EM map EM map + PDB 03/Nov/2021 Karel Kubíček 34 Dynamics in EM movie.7.mp4 State of the art in single particle EM – 1.25 Angstr. Diagram Description automatically generated Text Description automatically generated Nature, 2020 A picture containing diagram Description automatically generated A picture containing diagram Description automatically generated Skubnik, K; Sukenik, L; Buchta, D; Fuzik, T; Prochazkova, M; Moravcova, J; Smerdova, L; Pridal, A; Vacha, R; Plevka, P, 2021: Capsid opening enables genome release of iflaviruses. SCIENCE ADVANCES Computational + experimental methods 03/Nov/2021 Karel Kubíček 36 03/Nov/2021 Karel Kubíček 37 Diagram Description automatically generated Trends in Cell Biology, 13(3), 2003, 107-110 CLEM - Correlative light and electron microscopy 41592_2019_497_MOESM3_ESM.avi A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue, Nature methods, 2019 03/Nov/2021 Karel Kubíček 39 Villa et al. Current Opinion in Structural Biology, 23(5), 2013, 771-7 Diagram Description automatically generated 03/Nov/2021 Karel Kubíček 40 Villa et al. Current Opinion in Structural Biology, 23(5), 2013, 771-7 Diagram Description automatically generated Diagram Description automatically generated 03/Nov/2021 Karel Kubíček 41 A picture containing text, outdoor Description automatically generated This is done in several consecutive steps, when the ion beam is focus closer and close to the position of the desired lamella, with decreasing current to prevent undesired milling. Here you can see the milling process from he ion beam view and here from the electron beam view. The electron beam view is used to estimate thickness of the lamella, as thinner sample is more transparent for electrons and appears darker on the detector. 03/Nov/2021 Karel Kubíček 43 Villa et al. Current Opinion in Structural Biology, 23(5), 2013, 771-7 Graphical user interface Description automatically generated with low confidence CEITEC at Masaryk University 44 3 μm 3 μm 3 μm 5 μm 3 μm 2 μm On these pictures you can see what lamellae look like inside a TEM. Image A shows an unevenly milled lamella with dark longitudinal patches referred to as curtaining. Image B shows a partially crystallised lamellae as a consequence of imperfect plunge freezing. Image C then shows a well-polished and relatively thin lamella. Images D show the same lamella. D1 shows this lamella imaged in a SEM, and D2 and D3 in a TEM at different magnification. Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography, Elife 2015 elife-04889-media3.mp4 thylakoid tip convergence zone at the rim of the chloroplast cup Adobe Systems Diagram Description automatically generated EM Tomografie 03/Nov/2021 Karel Kubíček 47 Take home message: 1.Negative stain 2. 2.Single particle reconstruction 3. 3.Dynamics 4. 4.Tomography 5. 5.CLEM Adobe Systems Cryo-EM Grid Preparation - Vitrification using the Vitrobot Mark IV CEITEC at Masaryk University 49 Here are short videos from tomograms and sub-tomograms. The first video shows cell division, where we see the end of two splitting cells. The second video shows endoplasmic reticulum connected to the nuclear membrane. The last video shows an object that I unfortunately failed to identify. It consists of 12 filaments approximately 12-15 nm wide and has got a double membrane. Adobe Systems Define footer – presentation title / department 50 Obsah obrázku budova, bílá, vsedě, jídlo Popis byl vytvořen automaticky Obsah obrázku kobereček, jezdectví Popis byl vytvořen automaticky Obsah obrázku bílá, kámen Popis byl vytvořen automaticky Glucose-free (low polysome content) Standard (high polysome content) Heat shock 47° C (low polysome content) 200 nm 200 nm 200 nm A C B Images on this slide represent samples grown at different conditions with different polysome content Image A shows the standard sample with a mediocre amount of particles. Ribosomes are not distributed homogeneously but are rather clustered. This implies presence of polysomes. Image B shows a cell grown in glucose-free environment and there is a significant decrease of ribosomes. This results in a decrease in polysome content. Image C shows sample stressed by a heat shock. This resulted in a visual increase in the number of particles, which are distributed homogeneously, suggesting dissociation of polysomes and perhaps even ribosomes in favour of ribosomal subunits. A structure of the 80S ribosomes in situ Obsah obrázku různé, fotka, typ, mnoho Popis byl vytvořen automaticky Orientation #1 Orientation #2 Orientation #3 Orientation #4 New in situ model High–res structure #1 High–res structure #2 In my research, I managed to resolve a low resolution structure of the yeast 80S ribosome in situ. Column 1 shows my structure and column 2 and 3 show high-resolution ribosome structures previously resolved by other groups. Each of the row shows the same or similar orientation for each of the particles. Apart from low resolution, my structure has also some extra and missing electron densities, which was most likely caused by obvious inhomogeneity of the cellular sample. Diagram Description automatically generated with low confidence A picture containing chart Description automatically generated A picture containing application Description automatically generated Text Description automatically generated