Krios – Cryo-EM
Source: https://cryoem.iitm.ac.in/krios/ Parent: https://cryoem.iitm.ac.in/
KRIOS G4
Key Specifications
- Accelerating voltage: 300 kV
- Electron source: X-FEG (eXtended-field emission gun)
- Lens system: Triple-condenser design for beam parallelism and control
- Detectors: Falcon 4i, Ceta D
- Imaging Filter: Selectris Energy Filter
- Automated Cryo Autoloader supporting upto 12 grids
- Imaging mode: TEM and Diffraction
- Cryo-stage: ±70° tilt range
- Supports Fringe-Free Imaging (FFI) and Aberration-Free Image Shift (AFIS)
- Softwares: Thermo Scientific EPU for automated SPA, EPU-D and Tomography (Tomo5), Amira for visualization
- Offload Data Server: High performance server for automated data storage and fast transfer from detector
Applications
- Single Particle Cryo-EM (SPA)
- Micro-Crystal Electron Diffraction (Micro-ED)
- Cryo-Electron Tomography (Cryo-ET)
- High resolution TEM (HRTEM)
Applications
- Single-Particle Cryo-EM (SPA)
- Micro-ED
- Cryo Tomography
- HR TEM
Single-Particle Cryo-EM
It is a powerful and contemporary structural biology technique that allows one to determine the 3D structures of proteins and macromolecular complexes at near-atomic resolution without the need for crystallization. The practical limit on the molecular weight of the sample can be studied in SPA is ≥ 100 kDa. Other smaller proteins (50 – 100 kDa) are difficult as often different than conventional approach required and success depends on each sample.
The sample is vitrified on an EM grid and screened for thin ice and for good particles distribution within the holy support film. Thousands of particle images as dose fractioned movies are collected at high magnification from selected holes automatically. After, during image processing individual particles are boxed out computationally and aligned to get 2D class averages of similarly orientated molecules. Finally, particles from 2D class averages having high resolution secondary structural features are pooled together and aligned in 3D to obtain near-atomic reconstruction of the molecules. A model is derived from the final reconstruction.
3 D Electron Diffraction (3D ED) or Microcrystal Electron Diffraction (MicroED)
3D ED, also known as MicroED, is a powerful electron diffraction technique used to determine the atomic-level three-dimensional structure of crystals as small as a few hundred nanometers. It involves continuously rotating a tiny crystal under a parallel electron beam inside a transmission electron microscope (TEM) while capturing diffraction patterns. This allows rapid collection of a complete 3D dataset, which is then processed using established crystallography software to reveal high-resolution structural information. MicroED enables structure determination of samples too small or challenging for traditional X-ray crystallography, combining fast data collection with high resolution to analyze proteins, small molecules, and materials that are otherwise difficult to study.
Critically, MicroED is generally performed under cryogenic conditions using a cryo-TEM to minimize radiation damage to the delicate nanocrystals during electron beam exposure. Cryogenic cooling preserves structural integrity, allowing high-quality diffraction data to be obtained from beam-sensitive samples. This technique is particularly valuable because many commercially and medically important compounds can only be grown as nanocrystals or may undergo phase and polymorphic transitions during growth, making their analysis challenging.
Cryo-electron tomography (Cryo-ET)
It is a cutting-edge and high resolution imaging, structural biology technique that gives details of unique cellular events, virus-host membrane interactions, large biological assembly, target localization … etc in sub-nanometer resolution. Based on the type and thickness of the samples, they are either directly vitrified or milled in a cryo-FIB after high-pressure freezing to get thin section (~150nm) of target as a lamella. In this technique, the target of interest is imaged successively by tilting the stage ±60° in steps of 1-3° and collected images form a tilt-series. Later computationally the individual tilted images from a tilt-series are aligned and reconstructed in 3D to get a tomogram. Further, any repetitive structures present in a set of tomograms can be boxed out in situ as subtomograms and further averaged to get higher resolution (denoted as STA – SubTomogramAveraging).