SOLARIS Cryo-EM Facility provides access to two high-end cryo-electron microscopes dedicated to determining structures of biomacromolecules. In the experiments, we use single particle analysis (SPA), cryo-electron tomography (cET) and microcrystal electron diffraction (MicroED) techniques to analyse specimens at atomic resolution.
Research method description
Transmission Cryo-electron Microscopy (Cryo-EM) is an imaging method based on the elastic scattering of an electron beam passing through the studied material suspended in a vitreous ice layer. Electrons scattered during transmission create a two-dimensional enlarged projection of the sample on a detector. Due to the short wavelength of electrons (in the order of picometers), it is possible to achieve a near-atomic resolution of the measurement.
Proper sample preparation is crucial for cryo-EM studies. Samples are frozen in amorphous ice by rapid immersion in liquid ethane. This prevents the crystallization of water molecules and consequently preserves the native structure of the sample molecules. During imaging in a cryo-electron microscope, the sample is constantly cooled to the temperature of liquid nitrogen to preserve ice in the amorphous state and also to reduce radiation damage to the biological particles.
The three-dimensional structure of macromolecules is reconstructed using backprojection from thousands of micrographs of individual molecules, showing their two-dimensional projections in various, random orientations. Importantly, unlike X-ray crystallography, the cryo-EM technique does not require crystallization of the sample prior to the measurements.
SOLARIS Cryo-EM Facility founding & operation
The cryo-EM Facility at SOLARIS National Synchrotron Radiation Centre (NSRC) in Kraków, Poland, was established in October 2018 as a result of a joint initiative of a consortium comprised of 18 best Polish scientific institutions and R&D organizations conducting research in the field of structural biology. The consortium that helped in the Facility’s foundation operated under the leadership of dr hab. Sebastian Glatt (Malopolska Center of Biotechnology, Jagiellonian University, Kraków, Poland) and Prof. Marcin Nowotny (International Institute of Molecular and Cell Biology, Warsaw, Poland). SOLARIS Cryo-EM Facility was officially opened on the 1st of March 2019 during the first Cryo‑EM Consortium Meeting.
SOLARIS Cryo-EM Facility started user operations in September 2019, when the Titan Krios G3i cryo-TEM became available for the first time during the SOLARIS Autumn Call for proposals. SOLARIS Cryo-EM Facility has become a fully operational centre for cryo-EM studies, enabling scientists to perform state-of-the-art basic and applied research in a number of fields, e. g. life sciences, biotechnology, chemistry, and materials science.
At present, SOLARIS Cryo-EM Facility provides access to two high-end cryo-electron microscopes, i.e. Krios G3i and Glacios (both Thermo Fisher Scientific) fully dedicated to measurements at cryogenic temperatures.
|Cryo-Electron Microscope||Krios G3i Crio-TEM||Glacios Crio-TEM|
|Source||X-FEG (high-brightness field emission gun)|
|Accelerating voltage||300 kV||200 kV|
|Cryo-autoloader||Automated and contamination-free loading of cassettes (up to 12 Autogrids)|
Automatic condenser, objective and SA apertures
|Three-condenser-lens system for automated, continuous, and parallel sample illumination||Symmetric constant power objective lens (minimize image aberrations and lens hysteresis during mode switching)|
|Stage||Computerized 4-axis specimen stage with ±70-degree alpha tilt|
|Cryo-stage with single axis holder for optimized stability and drift performance||Cryo-stage with single axis holder|
Rotation-free imaging with changing magnification
|FFI (fringe-free imaging) Enhanced throughput with multiple image acquisitions per grid hole||-|
|Software||Thermo Fisher Scientific:
EPU software for single particle analysis (SPA) screening and data acquisition
|-||EPU-D software for microcrystal diffraction acquisition (micro-ED)|
Gatan K3 BioQuantum Direct Electron Detector
Falcon 4 Direct Electron Detector Thermo Fisher Scientific
|Energy filter||Gatan BioQuantum Energy Filter for contrast enhancement
|Other options||Thermo Fisher Scientific Phase Plate Solution for contrast enhancement||-|