Structural Biochemistry/Proteins/Cryo-Electron Microscopy
Cryo-Electron Microscopy specializes in interpreting and visualizing unstained biological complexes such as viruses, small organelle, and macromolecular biological complexes of 200 kDa or larger preserved in vitreous (i.e. glassy or non-crystalline) ice. The basic goal is to compare other electron microscopy techniques to use cryo-fixation to rapidly freeze the biological sample so as not to destroy its aqueous enviornment. This avoids ultrastructural changes, redistribution of elements, and washing away of substances. Specimens frozen in vitreous ice show a structure similar to the liquid state, or the native state. The near native imaging conditions allows three dimensional reconstruction of the cellular machinery. Using state of the art computer controlled, automated microscopes, image reconstruction software, and visualization tools, sub-nanometer resolution structures of large biological complexes can be achieved. In Cryo-Electron Microscopy, an electron beam, a stream of high energy particles bombards the sample. The image that is viewed is a result of the interaction of the sample with this beam. Most of the electrons that form the high resolution image appear due to elastic scattering, where only their trajectory has been changed, but their energy is unaffected. However, a small fraction of the electrons transfer some of their energy to the sample. This energy accumulates and can break apart molecular bonds, destroying the sample after some time. Therefore, for high-resolution imaging, low dose parameters require that the area to be imaged is not exposed until the picture is actually taken.
Cryo-electron microscopy can be performed by various methods of specimen preparation, two popular methods use thin film and vitreous sections of biological material. The thin film method requires biological material to be placed on an electron microscopy grid and is rapidly frozen close to liquid nitrogen temperatures. Larger samples (vitreous sections) can be vitrified by different methods including high pressure freezing. These samples can then be cut thinly and placed on the electron microscopy grid, similar to the thin film. These samples must remain at liquid nitrogen temperature to undergo the high vacuum and are exposed to the electrons.
One branch of Cryo-electron microscopy is Cyro-Electron tomography (CET). Cyro-electron tomography is performed at cryogenic temperatures as is cryo-electron microscopy; CET constructs a 3D sample from 2D images.
Uses for Cryo-Electron MicroscopyEdit
Cryo-electron microscopy is used in a variety of fields. Nanoparticle research relies heavily on electron microscopy for the visualization of small particles. Pharmaceutical companies doing drug research utilize electron microscopy to help predict the behavior of drugs and biological matter. In the case of pharmaceutical research a 3D visualization is extremely useful and at cyro-electron microscopy proposes the least damage to the sample to obtain a usable image.
Advantages in using Cryo-Electron MicroscopyEdit
- Allows the examination of native and hydrated structural features of the biological sample. The sample is always in solution and never comes into contact with an adhering surface. Therefore, the shape that is observed is the true shape of the hydrated molecule in solution and has not been distorted by attaching itself and flattening against the supporting film.
- Provides good preservation of biological structure in the microscope vacuum.
- There are no stains or chemical fixatives to distort the sample. When stained, the sample can be damaged in many ways, such as flattening and twisting.
- When the sample adheres to the carbon grid, it could stick in a preferential orientation. If this happens, then information will be missing from the final image set (a missing cone), and the resolution of the calculated model in that direction will be absent.
- Provides a 2-5 fold reduction in radiation damage compared to similar sugar-embedded or freeze-dried samples at room temperature. The reason behind this is thought to be from decreasing the temperature-dependent rearrangement or diffusion of fragments resulting from bond-fracture. In the solid frozen state, rearrangement or diffusion is decreased and the protein conformation is more likely to be maintained up to higher levels of irradiation.
- Can observe contrast between nucleic acids, proteins, and lipids to be distinguished.
- Enables one to control the chemical environment so that examination of different functional states of molecules is possible.
Disadvantages in using Cryo-Electron MicroscopyEdit
- Very low signal to noise ratio. Biological macromolecules are normally made up of carbon, hydrogen, oxygen, and nitrogen. The electron absorption of such molecules is very low. As a result, image contrast is also very low and it is hard to detect features when dealing with just a few images.
- Difficult to obtain images from tilted specimen. The ice cross section of a tilted frozen sample is too thick to yield good images.
- Charging is more widespread when imaging a tilted frozen sample.
- More time consuming to generate samples. However, this is generally not a big problem, especially once a working protocol is designed and good samples are readily available.
- If vitreous ice cannot be easily formed, the resulting cubic ice absorbs electrons very easily and the frozen sample is basically worthless.
- Sample must be maintained at less than 135 degrees Celsius.
Preparation of Frozen-hydrated Biological SpecimensEdit
The following are some general procedures for preparing frozen-hydrated biological specimens: 1)Development of a thin layer of the biological specimen. 2)Rapid cooling of the specimen to the vitreous state. 3)Transfer the specimen to the electron microscope without rewarming above the devitrification temperature. 4)Observe the specimen below the devitrification temperature with an electron dose that is low enough to preserve the structure of the sample.
Tomography uses the effects and differences that waves of energy have on a solid object to produce a three dimensional image of the internal structure. Cryo-Electron Tomography is a branch of Cryo-Electron Microscopy in which two dimensional projections of a frozen sample, at cryogenic temperatures, are recorded and used to reconstruct a three-dimensional structure by computed back projection. This is done using a transmission electron microscope to take successive images of a sample while tilting the sample around an axis. The “projection theorem” states that a 3D object can be retrieved from its projections along different directions. So to obtain a 3D description of an object, it must be projected along different directions; this is achieved by incrementally tilting the specimen. Due to the limitations with the transmission electron microscope (TEM), the specimen can only be tilted to +- 60-70 degrees, and not to 90 degrees which would be necessary to retrieve all the 3D information about the specimen.
Cryo-ET is a very accurate way to determine the three-dimensional structure of a specimen because the rapid freezing of the object and cryogenic temperatures gives a good preservation of the structure and good time resolution of certain processes. For example, the rapid freezing of cells and tissues at a certain point in cellular processes can give a good understanding of the structure and activity of those cells and tissues at a certain point in time during that particular cellular process. This type of tomography aids in the learning of cells and their organelles at a more dynamic level. Each organelle of a cell is produced in a different color, in order to facilitate the viewing process. The cells are frozen in order for the cell to retain its original structure. Freezing such specimens is done by placing them on a grid, blotting them in a thin layer of water and emerging them into ethane before storing them into liquid nitrogen. The use of cyro-electron tomography involves the study of almost all specimens, such as viruses. This tool can be helpful in understanding the replication states of viruses, as well as, the individual structures that viruses can become. A recent study has been done on the spikes of the viruses and the various structures the spikes affect the virus. Today, cyro-electron tomography is used to help find a cure for cancer by assessing the building blocks of the protein, cadherins, which aid in blocking cancerous tumors for spreading throughout the body. The information obtained through Cryo-ET can aid in comprehending and understanding the structural basis, and therefore, the function of many cellular processes.
There are limitations with Cryo-ET. The main limitation is the thickness of the specimen. The specimen must been thin enough for it to freeze well and so that it can be properly collected with the TEM. If the specimen is too thick, it must be cut into thinner slices while the temperature is still very low, so that re-crystallization does not occur. There are a couple ways to obtain the images, one of the is by fixed tilt increments, and the other is by graduated tilt increments. Graduated tilt increments are more favorable, this is when the tilt increment is proportional to the cosine of the tilt angle. Another issue with Cryo-ET is radiation damage. To prevent radiation damage, the specimen should be imaged under low electron dose conditions, leading to a more limited resolution in the 3D image obtained, and it also limits the specimen thickness needed for Cryo-ET.
Icosahedral Reconstruction refers to the application of cryo-electron microscopy in elucidating the structure of particles with appropriate (icosahedral) symmetry. The high internal symmetry of icosahedral specimens makes it easier to determine the positions of symmetry elements, thereby decreasing the amount of images required to determine the 3D structure of the specimen. This may seem like an arbitrary and irrelevant solid to apply to microscopy, but in fact there is an enormous number of particles that contain such symmetry. Examples of icosahedral particles include the majority of human viruses, as well as some molecules such as dodecahydro-closo-dodecaborate ion (B12 H122-) and the buckminsterfullerene.
A number of virus structures have been predicted and subsequently experimentally determined through the use of icosahedral reconstruction. An icosahedron belongs to the high symmetry group Ih which contains 120 symmetry operations, possibly most unique being the six five-fold symmetry axes. The properties of this symmetry group are essential for the application of cryo-electron microscopy.
Helical Reconstruction is a method that takes advantage of Cryo-Electron Microscopy in order to develop a three-dimensional structure for certain "filamentous" biological structures. This method used the 2-D projection images from Cryo-Electron microscopy to produce these 3-d Images as long as there is helical symmetry. This method however cannot be applied to structures that contain "seams" or "pertubations". There is a new method known as asymmetrical helical reconstruction that can be applied to helical structures that contain "seams". Similar to conventional Helical reconstruction methods, Fourier transform images are used to produce the layer line data which are then used to produce the 3-d structures.
Helical reconstruction allows the formation of large groups by regular contacts of a single type of protein molecule. Helical symmetry can be found in filamentous viruses (e.g., Pf1), in the proteins of the actin, tubulin, or other cytoskeleton, or in the proteins that form 2-D crystals folded onto the surface of a cylinder, such as the acetyocholine receptor or CopA.
Basic idea of 2-D v.s. 3-D helical reconstruction: Figure1 and Figure2. link: http://www.nysbc.org/facilities/CEM/cryoem-generalinfo.html A look of the 2D and 3D images of protein by electron microscopy methods.
Electron crystallography is a form of microscopy that uses a beam of electrons to construct images of small solids such as proteins. This process is used to determine and predict the structure and arrangement of a protein from secondary structure crystals such as alpha helices or beta sheets based on electron scattering. It can be used to study both organic and inorganic matters, and also protein structures. Electron Crystallography complements X-ray crystallography in many ways but also succeeds where X-ray crystallography fails. For example, X-ray crystallography study requires the quaternary structure of proteins which is often hard to attain than secondary structures. Electron Crystallography presents a problem in that it can cause radiation damage to the proteins under analysis. This hinders the range and function of the microscopy process. In order to reduce radiation damage, cryofixation, in which the imaging takes place in very low temperatures such as that of liquid nitrogen, is implemented. This resource is especially valuable when a specific protein is easily denatured or damaged by the electrons from the microscope.
A crystal structure determination includes two steps: ‘solving’ which finds a model of the heaviest atoms within about 0.25 Å using EM-images; and ‘refine’, which finds all atoms within about 0.02 Å using Selected Area Electron Diffraction or Convergent Beam Electron Diffraction data.
The use of electron diffraction in order to study the structures of crystals began in Moscow in 1937-1938 among a group of scientist led by Pinsker and Vainshtein. Their study used their own electron diffraction cameras that had relatively low acceleration to record electron diffraction data of different materials. From this data, they were able to locate hydrogen atoms in crystal structures which can not be done using X-ray diffraction. In order to solve unknown structures, phase information is needed which was first introduced by Hauptmann and Karle in 1953 called the "direct methods". Combining the use of direct phasing methods with modern day computers, electron crystallography has made significance advances in structure determination of crystals and other molecules.
There are two different electron diffraction techniques: 1)Selected Area Electron Diffraction (SAED)which requires near kinematic condition and applies for unit cell dimension >10 Å and for thin specimens <200 Å; 2)Convergent Beam Electron Diffraction (CBED)which makes use of dynamical effects and applies for unit cell dimensions <10 Å and for thick specimens >200 Å
Why electrons? Electrons are used in favor of X-rays because it is 10^4-10^5 times stronger interaction with matter compared with X-ray; and their phases are present in high resolution electron microscopy images.
There are some key advantages of electron crystallography compared to X-ray crystallography. One of these advantages is that electron crystallography can analyze much smaller crystals. This is because electrons interact more dominantly with matter than X-rays do. Another advantage is that electron beams can be focused by magnetic lenses to create an image while X-rays cannot form an image. Because the mechanism by which electrons interact with matter is based on the electrons detecting potential distributions in crystals compared to the mechanism of X-rays which depends on the X-rays detecting electron density distribution, electron crystallography can be used in certain situations that X-ray crystallography cannot. For example, the oxidation states of atoms in a crystal.
Using electron crystallography to determine structure is important due to the ability for a protein to be observed in its natural form. By utilizing electron crystallography, one can observe a protein in a lipid-protein bilayer in the structure that it is found in, thus allowing for better determination of function.
Single-particle electron microscopyEdit
The techniques used to reconstruct the 3 Dimensional images of the molecule from a collections of 2-D images is called electron microscopy. It presents to structural biochemists insight views in term of structural information of many biological molecules because of its easy-to-access features. In order to acquire 3D structure from this method, two requirement must met.
1. reasonable size of proteins to large macro-molecular assemblies without need to use crystals 2. the molecules must exist in many identical copies
The resolution produced by electron tomography have a low resolution and high noise. The main goal of the single particle electron microscopy is to determine the geometric relation between the collected projection images. In the year of 2008, scientist were able to made it possible to trace the backbone of the polypetide chains and build atomic models. Single Particles EM indeed have the capability to deliver structural information at near atomic resolution.
The 3D structure would be the result of these following steps:
1) Sample preparation. This is the step where sample is collected and place on (metal) plate to generate the best contrast. There are three technique used to prepare the samples for single particle EM. a) Negative staining : molecules are adsorbed to a continuous carbon film in which molecules are put into a metal plate by drying b) vitrification: sample is plunged into liquid ethane to preserve molecules in a native environment, it produced low contrast. (preserving them in a fully hydrated state)Vitrification is the best speciment preparation method, but not applicable to heterogeneous samples. c) Cryo negative staining: high contrast image immersed in high ionic strength of saturated ammonium molybdate solution. It is good to study the small and heterogeneous samples.
2) Particle picking. This is one of the most tedious processes of all because electron microscopists have to classify and separate particles according to their similarities in orientations; all these works are done by hand in order to achieve maximum efficiency. Having said that automated picking programs have been developed, but they failed to perform the task thank to the low signal-to-noise ratio. Result of this step is collection of small individual images of particles.
3) Generate initial model. Individual images collected from previous step are used to build a preliminary model. RCT is the primary method that is being used to generate initial models. Random Conial Tilt Reconstruction is being use throughout the process. Random Conical Tilt are the reconstruction of the 3 dimensional image one at high tilt angle and the other at untilt angle. The tilt angles allowd the testing samples to align in unique orientations. Please see the image at the right for the figure. Random Conical Tilt is common used in negatively or cryo-negatively stained speciments, in which works well with heterogenous particle solutions.
4) Refinement. The preliminary model is used to calculate for better alignment using Euler angles, in-plane rotated/unrotated shifts of particles. From refined data, a new 3D structure of a molecule is reconstructed. The risk of overrefinement happeneds when the testing negative temperature is applied.
Single-particle electron microscopy is having advantages ahead of electron crystallographic because its economic features. Unlike crystallographic, it does not require crystals – meaning samples don’t have to be pure - which take a lot more works to achieve. Another advantage is that single particle electron microscopy takes very little sample which always makes researchers happy.
One of the disadvantages of single particle electron microscopy, however, is that it is difficult to determine the resolution of density maps as well as their accuracy. Since there is no evidence of a method to check for accuracy, often the only thing that can be done is to repeat the process and compare results to previous results. Therefore, results can only be assessed on the basis of consistency rather than accuracy.
Electron crystallography has been used in the study of membrane proteins by analyzing arrays of samples called protein-lipid arrays. These arrays can be arranged in many different ways and offer many advantages and disadvantages. Two forms that have given the most useful density maps of membrane proteins are the two dimensional sheet like crystal arrays and the tubular like crystal arrays. These are so precise that they can reveal information about individual lipid molecules and the protein side chains due to averaging the many unit cells in the image of a sheet or tube like array enhancing the poor signal-to-noise ratio.
Over the last few decades there has been much advancement in the methods used to create these protein-lipid arrays both in tubes and sheets from detergent-solubilized purified proteins but no advancements in screens have been made like an easily manipulated robotic screen. Despite much research in the field they still lack the ability to quickly and reliably check the quality of the samples. Several laboratories have advanced the methods of analysis of these crystals making the process more efficient and more user friendly by enhancing the existing software.
The tubular crystals have not seen as extensive use as the sheet crystals even though their helical array symmetry allows for substantial advantages in determining structure. One image of a tube contains many different views of the same molecule which is enough to reconstruct it in three dimensions without the need for tilting. To correct for distortions tubes are processed in a similar way to sheets, where two repeat lengths are divided into shorter pieces and are then compared to a reference structure to determine the parameters needed to help identify the structure completely. This procedure traditionally uses Fourier-Bessel methods to assess the data, enabling them to analyze the extent of helical symmetry preservation and twofold symmetry perpendicular to the tube axis, which can correct for the focus changes at different levels of the structure. Another method has been developed that doesn’t employ the Fourier-Bessel methods and instead treats segments as strings of single particles. This alternative is becoming more popularly used for extracting structural information from poorly ordered helical polymers such as tubular protein-lipid crystals. This shows great potential for determining structures from tubes at the near atomic level of resolution.
Methods involved in electron crystallography include free-trapping to create different conformational states. To freeze-trap the specimen, the electron microscope grid is placed into liquid nitrogen-cooled ethane, which cools the specimen rapidly enough that thus allows for the trapping of a structure of a lipid-protein array which has a life-time of a millisecond or longer. The freeze-trapped protein can be activated through light or an appropriate ligand. Recent developments of helium-cooled top-entry freeze-trapping has resulted in a more clear image for data collection, and hopefully would allow for the gating mechanism of the protein-lipid bilayer to be described in more detail.
Additionally, molecular tomography is used to explore proteins in their functional context. A three-dimensional picture of an entire scene is possible to create though taking images from a series of tilt views, therefore creating a better three-dimensional image.
It is a method that observing dynamic behavior of single molecule to determine mechanism of action at level of an individual molecule, and to identify, sort and compare subpopulation and substructure within cell. In order to characterize the dynamics of molecular structures, scientists look to real-time trajectories of individual molecule; and by observing many of them, a histogram of the dynamical properties over the population could be figured.
X-ray crystallography or NMR, in comparison to single-molecule methods, provides detail structural view but limit by static molecular view and ensemble average.
1. Single-molecule manipulation: In this method, molecules are attached to an external probe which exerts defined forces or torques on molecule in order to characterize their mechanical properties. This method is also called atomic force microscopy (AMF). Because cell is seen as a factory in which many processes are carried out by specialized machinery which converts the chemical energy into force, torque and mechanical work - of which the attached probe will now come in to detect the dynamics and mechanism. This method is recently used to study the folding and unfolding of RNA molecules and the enzymes that catalyze these reactions, and to study RNA polymerase.
2. Single-molecule detection: The molecule is tagged with a fluorescent label in two locations in the form of a “donor” and “acceptor” that can undergo fluorescence resonance energy transfer (FRET). The trajectory of molecules then can be watched regarding to a change in the intensity of the florescence of the probe or regarding to the change in FRET. Another name of this method is fluorescence method. . This is a powerful method to study dynamic behavior of molecules, their stability and track particles’ movements in and outside cell. This method is used to study, for example, the multiple interactions during translation by the ribosome.
Between the two mentioned above, the fluorescence detection method is preferable to researchers because it requires less elaborate and complex instrumentations, but the down side is the photons collected by instrument is limited.
- ↑ Jiang, Wen, Zongli Li, Zhixian Zhang, Christopher R. Booth, and Matthew L. Baker. "Semi-automated Icosahedral Particle Reconstruction at Sub-nanometer Resolution." Journal of Structural Biology . 136. (2001): 214-225. Print.
- ↑ Miessler, Gary. Inorganic Chemistry. 3rd ed. Dehli: Pearson Education in South Asia, 2007. Print.
- ↑ Fuller, S.D., and S.J. Butcher. "Three-Dimensional Reconstruction of Icosahedral Particles-The Uncommon Line." Journal of Structural Biology . 116. (1996): 48-55. Print.
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