MAX-PLANCKGESELLSCHAFT MAX-PLANCKINSTITUT FÜR BIOCHEMIE General introduction to three-dimensional cryo-microscopy Winter School on Structural Cell Biology Julio Ortiz Wolfgang Baumeister MPI of Biochemistry Dept. of Structural Molecular Biology Martinsried, Germany Brno, 9th February 2015 1 Three-dimensional cryo-microscopy A concept that encompasses leading methods in electron microscopy for three-dimensional structure determination of biological samples, from macromolecules to cells, in close to native conditions (vitrified samples). Cryo-EM: The challenges • Samples that have to be studied in their hydrated state to ensure structural preservation • Suitable sample thickness to obtain molecular resolution Vitrification converts liquid water into amorphous ice Sample Preparation .............. Advance Micromachining Advance 3D Correlation I ., ___ + ir~,~+. -J,,_ +^ w,^Uw «-^-»4-+^;«« poor imaging conditions • Low-contrast due to weakly scattennq......... „ . J ^ Optimize Instrumentation building blocks e.g. detectors & phase Plates • Sensitivity to ionizing radiation low dose methods Advance Automation TEM HISTORY: An Anecdote... *You know," Szilard told Gabor over at cafe table in 1927, now that it is possible to make electron lenses, __________ At smaller and smaller wavelengths, you would achieve much more detailed resolution than is possible with microscopes using light. Gabor and Szilard pondered his idea for a few minutes, then agreed it would serve no 12 . After all, you cou!d not put living matter into Hie kind of vacuum tube needed to control electron beams. Besides, they concluded, so much power would be focused in the 1 oil 11 o ^ p v s p \ I y\ (111V StIIJjpit. But as Gabor later realized, with that idle suggestion Szilard had grasped the possibility of an electron microscope at least a year before anyone else. And of the incinerated samplet Gabor later wrote, "Who would have dared to believe ihat ths cinder would preserve not only the structure of microscopic bodies but even the shapes of organic molecules?'* Gabor would be remembered years later as the inventor of holography, for which he received the 1971 Nobel Prize in physics ^5 William Lanouette. Genius in the Shadows, A Biography of Leo Szilard, the Man Behind the Bomb WHY?: Microscopy with electrons At smaller and smaller electron wavelengths, one can achieve much more detailed resolution than with light. The wavelength of an electron is given by the de Broglie equation: energy E [keV] wavelength A [A] velocity v [km/sec] 20 0.086 76000 40 0.06 107000 60 0.059 131000 80 0.042 152000 100 0.037 170000 300 0.019 233000 1000 0.009 282000 Transmission electron microscopes can resolve details down to 7 A! Some highlights in TEM history 1931 Technical University, Berlin 1897 JJ. Thomson 1924 Louis de Broglie 1926 H.Busch G.P. Thomson/ in j7 1 ' C.J. Davisson and L.H. Germer Knoll & E. Ruska 1936 Metropolitan Vickers 1938 B. von Borries & E. Ruska 1939 Siemens&Halske 1962 R. Casta ig n & L Henry 1982 P.T.E Roberts et al. E. Ruska/ 1986 G Binning and H. Rohrer Discovered the electron (as a 'corpuscte1) Predicted the wavelength for the electron A = h/mv Magnetic and electric fields act as lenses for electrons Observed the wave properties by electron diffraction Built the 1st electron microscope (EM) Manufactured the 1st industrial EM (EM1) Produced the 1st practical EM (Siemens) - 10 nm resolution Started their commercial EM production Built the 1st energy filter Used the 1st CCD's for EM Received the Nobel Prize for TEM and STM E. Ruska und M. Knoll: Die magnetische Sammelspule für schnelle Elektronenstrahlen. Z. techn. Physik. Band 12, 1931, 389-400 und 448 M. Knoll und E. Ruska: Das Elektronenmikroskop. Z Physik 78, 1932, 318-339. Electron Microscope - basics Electron Imaging TEM Image Plane Electron Gun Illumination System Computer-controlled Stage SPECIMEN Energy Filter & Camera Lucie etal.JCB (2014) 1 The "GUN" Thermionic Emission Field Emission (FEG) Condenser System 2 Condenser lens system 2 3 Condenser lens system Twin lens 3 Objective System Bio-Twin / Super-Twin / Ultra-Twin Phase Plates 4 The "STAGE" Micro-Mechanical Motorized Stages Piezo-Controlled Motorized Stages 5 Sample Holders Side-entry Holders Multispecimen Holders Negative Plates/Film 6 Electron Detectors CCD Cameras Direct Electron Detectors ln-column Energy Filters 7 Energy Filters Post-Column Energy Filters The Gun: Types of Emitters Thermionic Gun Field emission Gun (FEG) THE LENSES: Electron Optics Gun crossover ■ /Double Condenser system : /Aperture Specimen Objective lens Objective aperture & back focal plane Diffraction pattern Image Intermediate lens Second image Projector lens -Final image Soft iron pole pieces Copper coils Electrons are manipulated using electromagnetic lenses. The electron beam coming from the gun is focused and shaped with the help of condenser lenses and apertures. The objective lens and the projection system are used to obtain and magnify a diffraction pattern or the real image. Optical Aberrations spherical aberration chromatic aberration rsoh = CJ Objective Lens Defocus Spherical A b erration Phase contrast can be described by the Phase Contrast Transfer Function, which highly depends on the instrument and the imaging conditions FFT Electron-sample interactions Backscattered e- SEM X-rays Cathodoluminescence Auger e- X# / ..r Secondary e- ......• ...... Sample TEM Elastically scattered e (1-10°) Inelastically scattered e (<1°) Only the direction of the electron is changed Unscattered e- The energy and the direction of the electron is changed Inelastic Scattering The amount of inelastic scattering increases with specimen thickness. Inelastic Mean Free Path (MFP), which is a dimension that indicates the path-length inside the specimen wherein all electrons (statistically speaking) will have undergone one inelastic scattering event. . . •# Typical mean free paths are of the order of: 050-100 nm for 120kV 100-200 nm for 200kV 150-300 nm for 300kV. Inelastic Scattering ..& magnetic prism! Inelastic scattering Magnetic Prism Electrons with different energy loss 'Zero-loss' filtering Methods: Instrumentation Transmission Electron Microscope FEI™ Titan Krios Some Properties: • Electron Gun: FEG operated at 300 KeV • Automated & robotic sample transfer • Automated data acquisition/ remote controlled • Stable goniometer with capabilities for dual-axis tilt tomography Three Branches of Tridimensional Electron Microscopy Electron crystallography Single-particle analysis (SPA) Cryo-Electron Tomography (CET) Biological Sample 3 D" EM WOľkflOW One Dilemma in Cryo-ET Acquire as many images as possible (>= 100-200) Over the largest possible angular range (+/- 70°) At the lowest' possible dose (~ 50-100 e/A2) Radiation damage Dose: 50 e/Ä2 Dose = current density per unit area j (A/cm2) multiplied by the exposure time t (s) (C/cm2, or e/A2 e- = electrons) Electron Dose: Signal&Noise -► vitrified chromatophore vesicles in Rhodobacter sphaeroides (Falcon I) Low Dose Acquisition scheme Cryo-electron tomography Single-axis tilt Electron Cryotomography A tilt series is recorded in a TEM by rotation of the sample holder around one axis, usually ca. 120 degrees. Aim: 3D reconstruction of the imaged object. Missing Wedge Problem Cryo-electron tomography Dual-axis tilt Electron Cryotomography Two orthogonal tilt series from the same object are recorded in a TEM. Aim: To gain isotropic resolution for higher fidelity of macromolecular complexes recognition. Missing wedge Limited tilt range implies an unsampled region (information is missed) Fourier space Real space (Lucie, v.,et al, Annual Review of Biochemistry, 2005) "Cryo-Electron Microscope" concept NEW APPROACHES IN CORRELATIVE STUDIES OF BIOLOGICAL ULTRASTRUCTURE BY HIGH-RESOLUTION ELECTRON MICROSCOPY By Professor H. Fernandez-Morant M.D., Ph.D, Committee on Biophysics University of Chicago Chicago 37, Illinois Paper presented at Concept also in: Fernandez-Moran, H. (1965) PNAS ROYAL MICROSCOPICAL SOCIETY'S Celebration of the 11 Tercentenary of the Microscope in Living Biology" April 9, 1963 Bethesda, Maryland 445-451 "Cryo-Electron Microscope" concept These "cryo- electron microscopes", operating at temperatures of 1 to 4 degrees Kelvin, would embody the following significant features: (a) highly stable superconducting electromagnetic lenses, with very ripple-free magnetic fields of a persistent current in the optimum case; (b) operation in ultra-high vacuum and low temperatures resulting in decisive advantages of minimized specimen contamination, specimen damage and thermal noise; (c) optimum conditions for both low voltage (i.e. 1 to 10 kV) and high voltage electron microscopy. In addition, the use of high-efficiency image viewing (single-crystal fluorescent screens) and recording devices operating at optimum low temperatures would make it possible to use high-speed cinematography and stroboscopic recording (e.g. obtained through pulsed T-F emission from pointed filaments) for attainment of high temporal resolution combined with high spatial resolution. Complementary metal-oxide-semiconductor (CMOS) based detector (i.e, K2 Summit™) Methods: Instrumentation Direct Electron Detectors Direct Detection Cameras • Falcon (FEI™) • K2 Summit™ (Gatan INC). General Properties: • High spatial resolution • High Detective Quantum Efficiency (DQE) • Higher sensibility than a CCD camera. Less electron dose required. Dual-axis tilt tomography - FIB wedge £ coil BL21 Single-axis Tilt Tomogram A Single-axis Tomogram B 31 £ coli BL21 MtlA385-SecM Falcon Cryo-FIB: Yeast xy-slice from 3D-segmentation 3D reconstruction Cryo-FIB: Molecular architecture of NPCs From proteomic inventory to architecture S. Nickell, C. Kofler, A. Leis, W. Baumeister: Nature Reviews Molecular Cell Biology 7 (2006) 225-230 More information... Videos and Presentations: • "Getting started in cryoEM" G. Jensen's Lab: http://cryo-em-course.caltech.edu • Sjors Scheres' Lab at the MRC: http://www2.mrc-lmb.cam.ac.uk/groups/scheres/impact.html Book: Electron Tomography JOACHIM FRAXK