Cryo-Electron Microscopy

Cryo-EM of single particles or helical filaments

This method is used to determine the high-resolution three-dimensional (3D) structure of isolated protein complexes. Samples are flash frozen in a thin layer of vitreous ice and imaged at cryogenic temperatures by transmission electron microscopy (TEM), e.g., using the TFS Titan Krios microscope. The direct electron detectors (DEDs) of these instruments greatly increase the achievable image contrast, and allow ‘movie mode’ imaging. In this mode, a series of dose-fractionated images (i.e., movies that are containing 40 to 100 frames) of suitable  sample regions are recorded. The movie mode data collection allows sample drift to be detected and corrected. After the required alignment, the individual frames are merged to increase image contrast, i.e., to improve the overall signal-to-noise ratio.

The Falcon4 detectors employed by the DCI alternatively allow recording image data as a series of X/Y electron impact coordinates, together with the time point of the arrival of the electron. Such "electron event recordings" can later be interpreted by software and transformed into movies of variable pixel resolution and a variable number of frames for a movie.

Each sample region imaged contains many protein complexes oriented in different ways. Their images, i.e., projections in 2 dimensional space (termed 2D projections) are matched, classified and reconstructed in a procedure called ‘single particle analysis’ to obtain the 3D structure of the complex. Alpha helices and beta sheets can be visualized by this method, and, since the introduction of DEDs, atomic resolution is frequently achieved.

Sample Requirements for Single Particle or Helical Cryo-EM+-

A single particle cryo-EM project requires about 30 microliters of protein solution at a concentration of around 1 mg/ml. For a 100 kDa protein that would correspond to 10 micromolar concentration. For a 200 kDa protein it would correspond to 5 micromolar concentration.  Samples at lower concentration can sometimes be up-concentrated, but one has to pay attention to not also up-concentrate detergents if present.

The protein should be in a buffer that it is happy with. Salt concentrations should be below 200mM, otherwise the solution becomes rather dense for the electron beam and contrast is lowered in the electron microscope.  Mercaptoethanol or higher concentrations of glycerol often make problems for cryo-EM investigations and should be avoided.

The protein should be pure (An SDS-PAGE shows that >50% of the particles are the protein of interest), homogeneous (a SEC profile shows a symmetric narrow peak), and stable (the SEC profile looks the same a few hours later).

Cryo-EM workflow+-

As first characterization, negatively stained preparations on TEM grids should be prepared and investigated with a 120kV TEM. This can be done at the EMF at UNIL or at the CIME or the BioEM Lab at the EPFL. (For info on these partner facilities, please see on the front page of this website). Neg. stain TEM requires only 0.1 mg/ml protein concentration, and is often incompatible with phosphate buffers, as these tend to precipitate with the Uranyl Acetate stain.

If neg.stain TEM shows a suitable population of particles, cryo-EM grids have to be prepared by plunge freezing. For this, typically 3 microleters of sample solution at 1 mg/ml is given onto a glow-discharged fenestrated cryo-EM grid, blotted for a few seconds, and rapidly frozen to -196ºC by plunging into LN2-cooled liquid ethane. Grids are then inspected in a 120kV or 200kV cryo-EM instrument, which is available at the EMF@UNIL or the CIME/BioEMLab@EPFL or also at the DCI. 

If cryo-EM grids show a suitable density and distribution of randomly oriented particles that are freely suspended in thin vitrified ice, a larger set of dose-fractionated cryo-EM images can be recorded with a 300kV Titan Krios instrument at the DCI. 

A data collection session takes 24 hours or longer, depending on the biological question, target resolution, and the particle density and homogeneity. In some cases, a heterogeneous particle population is desired, to simultaneously capture various protein conformations within one data set.

Cryo-EM workflow for helical samples+-

Cryo-EM image processing+-

Recorded data will be computer processed during data collection on DCI computers, so that a first image processing results is available during data collection. Subsequent in-depth processing of recorded images, 3D structure reconstruction and atomic model building requires GPU computing hardware, certain software systems, and expertise, all of which is available at the DCI. Please contact us for more info.