10.2 Cryo-electron microscopy
5 min read•august 1, 2024
(cryo-EM) is revolutionizing . It freezes samples ultra-fast, preserving their natural state, and lets us see big, complex molecules in amazing detail. No need for tricky crystals or super-pure samples!
Cryo-EM shows us proteins and other biological stuff in action. We can see different shapes and how they move, even inside cells. It's perfect for studying membrane proteins and big molecular machines that other methods struggle with.
Cryo-EM Principles and Advantages
Cryo-EM Technique and Benefits
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- Cryo-EM rapidly freezes biological samples in liquid ethane (vitrification), preserving their structural integrity and allowing visualization in their native state
- Cryo-EM enables the study of large, complex, and heterogeneous biological systems without requiring crystallization or extensive purification
- The technique determines high-resolution structures at near-atomic levels (3-5 Å), providing detailed insights into macromolecular structure and function
- Cryo-EM is particularly useful for studying membrane proteins, which are difficult to crystallize and analyze using traditional X-ray crystallography methods
Visualization of Conformational States and Native Environment
- Cryo-EM allows the visualization of multiple conformational states of macromolecules, revealing their dynamic behavior and functional mechanisms
- The ability to study macromolecules in their native environment, such as within intact cells or organelles (in situ), is a significant advantage of cryo-EM over other structural biology techniques
- Cryo-EM captures the structural heterogeneity of biological samples, providing a more comprehensive understanding of their functional diversity
- The technique enables the investigation of macromolecular interactions and the assembly of large complexes (ribosomes, viruses) in their native context
Sample Preparation for Cryo-EM
Vitrification Process
- Vitrification involves the of biological samples in liquid ethane, preventing the formation of ice crystals and preserving the native structure of macromolecules
- Samples are applied to a grid, typically made of copper or gold, and excess liquid is removed by blotting with filter paper
- The grid is plunged into liquid ethane cooled by liquid nitrogen, forming a thin, layer containing the embedded macromolecules
- The concentration and purity of the sample are critical factors in obtaining high-quality cryo-EM data, as aggregation or contamination can interfere with image processing and structure determination
- The thickness of the ice layer must be carefully controlled to ensure full sample embedding and minimize the effects of electron scattering and absorption
Grid Preparation and Treatment
- Grid preparation involves selecting and treating suitable support grids for cryo-EM samples
- Grids are typically coated with a continuous carbon film or a holey carbon film to provide mechanical stability and reduce sample movement during imaging
- Glow discharge or plasma cleaning is often used to make the grid surface hydrophilic, improving the distribution and adherence of the sample
- The choice of grid material (copper, gold) and mesh size depends on the sample characteristics and the desired resolution
- Sample application methods, such as manual pipetting or automated dispensing, are optimized to ensure uniform distribution and minimize air-water interface interactions
Single-Particle Analysis in Cryo-EM
Particle Picking and Classification
- (SPA) reconstructs the 3D structure of a macromolecule from a large number of 2D projection images obtained by cryo-EM
- Particle picking identifies and extracts individual particle images from cryo-EM micrographs
- Particle picking can be performed manually or using automated algorithms that detect and select particles based on size, shape, and contrast
- Extracted particle images are classified and aligned to generate 2D class averages, representing distinct views of the macromolecule
- Classification sorts particle images into groups with similar orientations and conformations, improving signal-to-noise ratio and reducing heterogeneity effects
- Alignment determines the relative orientations of particle images and brings them into register to create a coherent set of 2D projections
3D Reconstruction and Refinement
- 2D class averages are used to generate an initial 3D model of the macromolecule, which is iteratively refined using projection matching and algorithms
- Projection matching compares 2D class averages with simulated projections of the 3D model to determine orientation and assign Euler angles to each particle image
- 3D reconstruction methods, such as weighted back-projection or iterative refinement, combine 2D projections into a consistent 3D density map
- The resolution of the final 3D reconstruction is assessed using Fourier shell correlation (FSC) and other validation metrics to ensure the reliability and accuracy of the obtained structure
- Refinement strategies, such as particle polishing, CTF correction, and beam-induced motion correction, are applied to improve the quality and resolution of the reconstructed density map
Applications of Cryo-EM
Studying Large Macromolecular Complexes
- Cryo-EM has been instrumental in elucidating the structures of large macromolecular complexes, such as ribosomes, viruses, and molecular machines, which are often difficult to study using other techniques
- Visualizing these complexes in their native states has provided insights into their assembly, function, and interactions with other cellular components
- Examples include the structure of the ribosome-translocon complex, the spliceosome, and the nuclear pore complex
- Cryo-EM enables the study of macromolecular complexes in different functional states, revealing conformational changes and dynamic interactions
- The technique has shed light on the assembly pathways and maturation processes of viral capsids, such as HIV and influenza viruses
Membrane Protein Structure Determination
- Membrane proteins, which account for a significant portion of the human proteome and are important drug targets, have been a major focus of cryo-EM studies
- Cryo-EM has enabled the determination of high-resolution structures of ion channels, transporters, and G protein-coupled receptors (GPCRs) in their native lipid environments
- These structures have revealed the molecular mechanisms of ion selectivity, gating, and ligand binding, guiding the development of targeted therapeutics
- Cryo-EM allows the study of membrane proteins in different conformational states, providing insights into their activation and regulation mechanisms
- The technique has been used to investigate the structure and function of membrane-associated complexes, such as respiratory chain supercomplexes and photosynthetic systems
Investigating Dynamic Biological Processes
- Cryo-EM is increasingly being used to study dynamic biological processes, such as protein conformational changes, ligand-induced activation, and macromolecular interactions
- Time-resolved cryo-EM techniques, such as flash-freezing or mixing-spraying, allow the capture of transient intermediate states and the reconstruction of conformational landscapes
- Examples include the study of ribosome dynamics during translation, the conformational changes of ion channels during gating, and the assembly of viral capsids
- Cryo-EM has been combined with other techniques, such as cryo-electron tomography (cryo-ET) and correlative light and electron microscopy (CLEM), to study macromolecular complexes within their cellular context
- Cryo-ET enables the 3D visualization of macromolecules and their interactions within intact cells or organelles, providing insights into their native organization and function
- CLEM combines fluorescence microscopy with cryo-EM to localize specific macromolecules within cells and guide the selection of regions for high-resolution structural analysis
Key Terms to Review (18)
3D Reconstruction: 3D reconstruction is the process of capturing the shape and appearance of real objects to create a three-dimensional model. This technique combines multiple images or data points from various angles, which are then processed to generate a detailed representation that can be analyzed and visualized. In scientific research, especially in structural biology, this method provides crucial insights into the spatial arrangement of biomolecules, enhancing our understanding of their function.
Cryo-electron microscopy: Cryo-electron microscopy (cryo-EM) is an advanced imaging technique used to visualize biological samples at near-atomic resolution while preserving their native state by freezing them in liquid ethane or propane. This method allows scientists to study the structure of macromolecules and complexes in their functional forms, bridging the gap between traditional electron microscopy and X-ray crystallography.
Electron detector: An electron detector is a device used to measure and analyze electrons that are emitted from a sample during techniques such as cryo-electron microscopy. This tool is critical for capturing high-resolution images of biological macromolecules and cellular structures, allowing researchers to visualize the fine details of samples at near-atomic resolution. The efficiency and sensitivity of electron detectors directly impact the quality of the resulting images in cryo-electron microscopy.
Electron diffraction: Electron diffraction is a technique used to study the arrangement of atoms in a material by observing the patterns formed when a beam of electrons is scattered by the sample. This method allows scientists to gain insights into the structure and properties of materials at the atomic level, making it an essential tool in fields like materials science and biophysics.
Image averaging: Image averaging is a computational technique used to enhance the quality of images obtained through methods like cryo-electron microscopy by combining multiple images of the same sample. This process reduces noise and improves resolution, allowing for a clearer view of the specimen's structure. It is particularly crucial in analyzing biological macromolecules, where individual particle orientations can lead to variability in image quality.
Joachim Frank: Joachim Frank is a prominent biophysicist known for his groundbreaking work in the development of cryo-electron microscopy (cryo-EM), a technique that allows for the visualization of biological macromolecules in their native state at high resolution. His contributions have revolutionized structural biology by providing researchers with tools to observe complex biomolecules, leading to significant advancements in understanding cellular processes and drug design.
Model fitting: Model fitting is the process of adjusting a mathematical model to best represent the data obtained from experiments or observations. This technique is crucial in various scientific fields as it allows researchers to derive meaningful interpretations and predictions based on empirical data, enhancing the understanding of complex biological structures and processes.
Phase contrast: Phase contrast is a microscopy technique that enhances the visibility of transparent specimens, such as living cells, by converting phase shifts in light waves into variations in brightness. This method allows researchers to observe fine details in samples without the need for staining, preserving the natural state of the specimen.
Protein complexes: Protein complexes are assemblies of multiple protein molecules that interact to perform specific biological functions. These structures can vary in size and complexity, often forming through non-covalent interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions. They play critical roles in cellular processes, including signaling, catalysis, and structural support, making their study essential in understanding molecular biology.
Radiation damage: Radiation damage refers to the detrimental effects caused to biological tissues and structures when exposed to ionizing radiation. This type of damage can lead to alterations in molecular structures, including DNA, proteins, and lipids, which may result in cellular dysfunction or death. Understanding radiation damage is critical in fields like cryo-electron microscopy, where minimizing such damage is essential for accurate imaging of biological samples.
Rapid freezing: Rapid freezing is a technique used to quickly solidify biological samples by exposing them to extremely low temperatures, usually in a matter of milliseconds. This method preserves the native structure of biomolecules and cellular components, minimizing ice crystal formation that can disrupt delicate structures. This preservation is critical for accurate imaging and analysis in techniques like cryo-electron microscopy.
Resolution assessment: Resolution assessment is the process of evaluating the ability of a microscopy technique, such as cryo-electron microscopy, to distinguish between two closely spaced objects or features in a sample. This term is critical as it determines the clarity and detail that can be observed in the resulting images, impacting the overall understanding of biological structures at the molecular level. In cryo-electron microscopy, achieving high resolution is essential for visualizing complex biomolecular assemblies and understanding their functional mechanisms.
Richard Henderson: Richard Henderson is a prominent biochemist known for his pioneering contributions to the field of cryo-electron microscopy, particularly in the determination of high-resolution structures of proteins and other biological macromolecules. His work has greatly advanced the technique, making it a crucial tool in structural biology for visualizing complex molecular structures that were previously difficult to analyze.
Sample stability: Sample stability refers to the ability of a biological or chemical sample to maintain its structural and functional integrity over time under specific conditions. This concept is crucial in ensuring that the sample does not degrade, denature, or undergo any unwanted changes that could affect experimental outcomes. In the context of cryo-electron microscopy, sample stability is paramount as it directly influences the quality of the images obtained and the accuracy of structural analyses.
Single-particle analysis: Single-particle analysis is a powerful technique used in structural biology to study the three-dimensional structures of individual macromolecules, such as proteins and viruses, without the need for crystallization. This method provides detailed insights into the conformational states and dynamics of these molecules, making it possible to visualize them at high resolution using imaging technologies like cryo-electron microscopy.
Structural Biology: Structural biology is the branch of biology that focuses on the molecular structure of biological macromolecules, including proteins, nucleic acids, and complex assemblies. It aims to understand how the three-dimensional shape of these molecules relates to their function, and employs various techniques from disciplines such as biochemistry, biophysics, and computational biology to investigate these relationships.
Transmission Electron Microscope: A transmission electron microscope (TEM) is a sophisticated imaging tool that uses a beam of electrons to create high-resolution images of thin specimens, allowing scientists to observe the internal structure of cells and materials at a nanometer scale. This type of microscope is essential for studying biological samples and materials science, as it provides detailed information about the arrangement and composition of atoms within a sample.
Vitreous ice: Vitreous ice is a non-crystalline, amorphous form of ice that is produced when water is cooled rapidly to below its freezing point without the formation of crystalline structures. This unique state of ice retains a glass-like quality, which makes it significant in various scientific applications, particularly in cryo-electron microscopy, where it preserves biological specimens in their native state without damaging them through crystallization.