Most you will associate EM with pictures like the one shown to the left. Courtesy Dr. Marc Paypaert, Yale Univ CCMI of Molecular EM-New Frontiers Tubulin a/b-heterodimer from 2D-crystals Coutesy Nogales&Downing Tubulin model fitted into helical reconstruction of a microtuble Coutesy Nogales,Downing&Milligan Jan 13,1968 the birthday of molecular EM Incidently, the discovery of how EM-images can be interpreted occurred at a time that saw the first X-ray crystal structures emerge. …and back then, X-ray crystallographic structures looked similar to what EMstructures look like today. …so, how comes that X-ray crystallography took off like a “rocket” while EM was a “sitting duck”? EM faced a number of unique challenges that needed to be overcome before it could become a generally useful tool for analyzing the structure of biological specimen. First: EM requires a vacuum - an environmental constraint that is incompatible with unprotected biological material. Electron beam specimen negative stain image Historically, specimen were protected against the vacuum by negative staining procedures. These protocols exploit that salts of heavy metals are relatively insensitive towards electrons and form a stable “cast” around the molecules when dried down. Salts such as uranylacetate or phosphotungstic acid titrated to neutral pH, vanadates and molybdates have and are still being used. The granularity of the stain limits resolution to about 15Å. Also, no internal detail is discernible because what is observed is the stain-exclusion pattern Finding a way around the use of negative stain was paramount for making any more progress towards meaningful EM-work on biological samples A simple example: Liposomes 4,400x liposomes negative stain Electron beam image Negative Stain: image shows stain exclusion pattern No internal detail/features are visible 26,000x Sample Vitrification If water is frozen very rapidly, it turns into an amorphous, solid state known as vitreous water. In this state, the water molecules are disordered and form a transparent “glass-like” solid around the biological sample. Hence, we directly observe the biological molecule and its internal features when imaging in the frozen hydrated state. Piston of plunger forceps EM-grid liquid ethane vitrification occurs at 105 ˚C/sec liquid nitrogen Negative Stain liposomes negative stain Frozen-Hydrated vs Electron beam image Negative Stain: image shows stain exclusion pattern No internal detail/features water image Frozen Hydrated: embedding medium is “transparent” Internal detail/features are revealed The Destructive Power of Electrons After 0.2 sec 1 sec exposure “In theory, there is no difference between theory and practice. In practice, there is” Frozen hydrated Negatively stained Particle picking from images of frozen hydrated samples can be challenging… Low contrast imposes certain minimum requirements on the state of the sample Structure determination of small molecules requires crystalline samples, known as 2D-crystals That is, in the case of 2D-crystals we can use a crystallographic approach towards structure. The advantage of having images is that there is no “phase problem” in electron crystallography! Electron Microscopy of Biological Specimen APPROACHES Crystalline Non-Crystalline 2D-crystals Single layer of molecules ordered in xy plane Examples: BR, LHCII, AQP1 and tubulin H+-ATPase, PSII, rhodopsin, gap junctions Easiest approach to get resolution Resolution range: 3-20Å to helical Examples: actomyosin complex, decorated lipid nano-tubes, RNA polymerase Resolution range: 20-35Å icosahedral Examples: hepatitis B core antigen (~7.5Å) Resolution range: 7-40Å high helical-crystals “2D-crystal wrapped onto a cylinder” Examples: AChR, Ca2+-ATPase Not so easy to get to high resolution, but AChR is currently at ~4-4.5Å Resolution range: 4-35Å single-particle Examples: Complex I, GroEL (7.5Å), latrotoxin dimer (~240kD), ribosome (8Å, phasing) Resolution range: 7-40Å tomography Examples: mitochondria, muscle Resolution range: >40Å Common to all except for tomography: Averaging! insect flight Use of noisy images require us to average many individual particle images. Lets briefly dwell on averaging and what it means…. The final structure will only reveal the features that are shared by all particles. The need for averaging also contains a potential danger. Can you see what it is? The presence of unrelated objects, conformational heterogeneity, or particles with slightly different compositions (e.g of subunits) all can invalidate the final result and/or complicate the analysis Source: “Biophysical Electron Microscopy” (Academic Press) Chapter 8 p:300 DIMENSIONS TISSUES CELLS MOLECULES cm-mm mm nm 10-2 - 10-3 10-6 10-9 ATOMS > nm [Å] 10-10 Light-Microscopy NMR, X-ray Electron-Microscopy Flowchart for a Typical cryoEM-Project Purified Specimen Sample Preparation Thin, Vitrified Specimen Cryomicroscopy Structure-Function Relationships Map Interpretation/Model Building Fitting of High Resolution Structures 3D Density Map Image Selection Digitization Image Processing & 3D-Reconstruction Micrographs Adapted from Baker and Henderson, International Tables of Crystallography Vol F Core Facility for Electron CryoMicroscopy Instrumentation 120keV Tecnai 12 TEM for screening & electron diffraction 200keV Tecnai F20 Field Emission TEM for high resolution imaging • Accessory equipment necessary to perform cryo-EM & to allow high throughput data collection on both systems • Optical bench • High resolution image scanner • DEC Alpha ES-40 server and workstations for digital image processing • SGI workstations for molecular graphics (as part of Bioinformatics Core) How to get started? 1. Identify specimen: size (the bigger the better, preferably >500kDa, but can do almost anything if not afraid of trying to crystallize sample….) quality: single protein? Yes: >90% pure No: substoichiometric subunits? stability: the more stable the better. Avoid glycerol if possible…. quantity: negative stain (few µg), cryoEM: comassie stainable (if possible) 2. Get excited and contact either Vinzenz (5-5652, vinzenz.unger@yale.edu) or Fred (5-5773, fred.sigworth@yale.edu to arrange for a quick sample evaluation and/or discuss your specific case. http://cellserv.med.yale.edu/imaging/ccmi/cryo_electron.html 3. Join the user group and have one of your students or postdocs trained. 4. Publish structure…. How long will it take? Hard to predict, but can potentially be long-term (1-3 years). May consider doing a reconstruction in negative stain first (this is a good idea anyway), which usually takes a lot less time. How much will it cost? Training: free of charge, provided you use the facility after training is completed. Usage: current fee is set at $30/hr (--> ~$5,000 if you plan to use the EMs for 3hrs a week). If the project transitions to cryoEM you need to commit ~$10,000 per sample. There are safeguards build into the policies of the facility that account for unforeseen hardship = in short: don’t worry - come on in and enjoy!