The Atomic Resolution Cryogenic Transmission Electron Microscopy (TEM) suite supports EMSL’s mission to improve our fundamental understanding of biological and environmental systems through:
- De novo structure determination of purified soluble or membrane protein complexes and protein/DNA/RNA complexes greater than 50kDa total assembled molecular weight
- High-resolution imaging of isolated organelles, microcompartments, whole cells, or more complex biosystems in normal and environmentally stressed conditions
- Morphological studies using electron tomography on unicellular and filamentous organisms, biofilms, plant-microbe-fungi, or host-viral/phage interactions
- Structure determination from microcrystals and nanocrystals of natural products, metabolites, or proteins
Two access proposal mechanisms:
Access to cryo-TEM with other capabilities at EMSL is gained via standard proposal submission to standard EMSL User Proposal calls with respective due dates.
Access to cryo-TEM ONLY is gained via proposals for the use of only cryo-EM/ET or micro-ED; may undergo an accelerated review and access process with no specified proposal due date. To learn more, contact James Evans.
The Krios Cryo-TEM can solve the structure de novo of proteins, macromolecular complexes, and small molecules. Structure resolution ranges from angstrom to nanometers, depending on sample type, size, and homogeneity. Three different sample workflows are available: cryo-EM Single Particle Analysis; micro-Electron Diffraction; and cryo-Electron Tomography. Krios supports the Structural Biology Integrated Research Platform.
When combined with access to the Aquilos 2 cryo-Focused Ion Beam (FIB)/Scanning Electron Microscope thick samples can be thinned to make them compatible with imaging on the Krios Cryo-TEM instrument. The thinning can be performed by lamella generation of samples directly on the grid, or site-selective lift-out where a region of a sample is plucked out of a tissue or thick sample and then moved and attached to a TEM grid for thinning and downstream imaging. By thinning samples to ~300-500 nm in thickness, this approach makes it possible to perform high-resolution electron tomography and sub-tomogram averaging to visualize the architecture of cells or perform micro-electron diffraction from thick crystals. This supports both the Rhizosphere Function and Cell Signaling and Communications Integrated Research Platforms.
Supporting the Biogeochemical Transformations Integrated Research Platform, these resources can provide high-resolution 2D and 3D morphological characterization of carbon black, sea spray aerosols, natural minerals, and organic/inorganic interfaces.
Krios G3i Cryo-Transmission Electron Microscope (Thermo Scientific)
- Autoloader can load 12 samples at a time for screening.
- K3 direct electron detector provides state-of-the-art images.
- Phase plate and energy filter can image thicker, whole cells for electron tomography.
- Batch and semi-automated workflows for single particle analysis, micro-electron diffraction, and tomography data collection.
- Real-time dedicated image processing pipelines provide rapid data quality monitoring and 3D reconstructions
Aquilos 2 Cryo-Focused Ion Beam/Scanning Electron Microscope (Thermo Scientific)
- This platform prepares samples for Krios imaging by thinning samples that are conventionally too thick.
- Thinning is possible with lamella generation or site-selective lift-out.
Leica EM/GP Vitrification System
- System performs vitrification of samples <10µm in thickness.
- Samples adhered to standard 3 mm diameter TEM grid.
- This is the primary system for vitrifying samples of purified macromolecular complexes, micro- and nanocrystals, and unicellular organisms.
Leica HPM100 System
- Performs high-pressure freezing and vitrification of samples <200µm in thickness.
- Runs samples frozen in copper hats with dimensions of 1.5 mm diameter and 200 µm height for cryo-ultramicrotomy or thinning with the Aquilos 2 cryo-FIB/SEM and downstream imaging on the Krios cryo-TEM.
Tips for success
Critical aspects of sample preparation include:
- All biological samples are embedded in vitrified ice without any chemical fixative.
- Attainable resolution for single particle 3D structure determination is highly dependent on sample homogeneity, particle distribution, assembly state, complex size, and ice thickness. Providing highly biochemically pure and well characterized samples is the ideal entry point to the workflow.
- Screening for optimal freezing conditions can take as long or longer than a data collection run. Every protein is different, and vitrification can be impacted by buffer composition, grid and protein electrostatics, local humidity, temperature, etc.
- De novo structure determination of small molecules and short peptides is possible with Micro-ED. However, Micro-ED of protein crystals requires the target protein to have significant homology to an already known structure, so molecular replacement approaches can be used for phasing and structure solution.
- 3D resolution for electron tomography is highly dependent on sample thickness, sample tolerance to electron dose, and desired field of view. Most tomography datasets will be limited to 0.8-3 nm resolution.
- Cell environments in standard growth or culture conditions are preserved until the moment of plunge-freezing, which occurs on a millisecond timescale.
- Ideal samples for tomography are less than 0.4 microns thick, either naturally or thinned by cryo-FIB or ultramicrotomy.
Compatible science samples:
- Biosystems in normal and environmentally stressed conditions
- Unicellular and filamentous organisms, cell cultures, and biofilms
- Plant-microbe-fungi interactions under different environmental conditions
- Host-viral/phage interactions
- Microcrystal and nanocrystals of natural products, metabolites, or proteins
- Isolated organelles or microcompartments
- Purified soluble or membrane protein complexes and protein/DNA/RNA complexes greater than 50kDa total assembled molecular weight