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Topic Name: Using New Technique Purdue University Researchers Take a Big Step in Examination of Small Structures

Category: Optical imaging

Research persons: Purdue Research Team

Location: Purdue University, United States

Details

A team led by a Purdue University researcher has achieved images of a virus in detail two times greater than had previously been achieved.

Wen Jiang, an assistant professor of biological sciences at Purdue, led a research team that used the emerging technique of single-particle electron cryomicroscopy to capture a three-dimensional image of a virus at a resolution of 4.5 angstroms. Approximately 1 million angstroms would equal the diameter of a human hair.

"This is one of the first projects to refine the technique to the point of near atomic-level resolution," said Jiang, who also is a member of Purdue's structural biology group. "This breaks a threshold and allows us to now see a whole new level of detail in the structure. This is the highest resolution ever achieved for a living organism of this size."

Details of the structure of a virus provide valuable information for development of disease treatments, he said.

"If we understand the system - how the virus particles assemble and how they infect a host cell - it will greatly improve our ability to design a treatment," Jiang said. "Structural biologists perform the basic science and provide information to help those working on the clinical aspects."

A paper detailing the work was published in the Feb. 28 issue of Nature.

Roger Hendrix, a professor of biological sciences at the University of Pittsburgh, said what is learned about viruses can be applied to many other biological systems.

"Understanding the proteins that create the structure of a virus gives us insight into the tiny biological machines found throughout our bodies," he said. "Getting to 4.5 angstrom using this technique is a watershed of sorts because it is the first time we can actually trace the polypeptide chain - the backbone of proteins. Now we can see the tiny gears and levers that allow the proteins to move and interact as they carry out their intricate biological roles."

The imaging technique, called cryo-EM, has the added benefit of maintaining the sample being studied in a state very similar to its natural environment. Other imaging techniques used regularly, such as X-ray crystallography, require the sample be manipulated.

"This method offers a new approach for modeling the structure of proteins in other macromolecular assemblies, such as DNA, at near-native states," Jiang said. "The sample is purified in a solution that is very similar to the environment that would be found in a host cell. It is as if the virus is frozen in glass and it is alive and infectious while we examine it."

In addition to Jiang, Matthew L. Baker, Joanita Jakana and Wah Chiu from Baylor College of Medicine, and Peter R. Weigele and Jonathan King from Massachusetts Institute of Technology worked on the project, which was funded by the National Institutes of Health and the National Science Foundation.

The team obtained a three-dimensional map of the capsid, or protein shell, of the epsilon15 bacteriophage, a virus that infects bacteria and is a member of a family of viruses that are the most abundant life forms on Earth, Jiang said.

Other methods of determining the structure could not be used for this family of virus. None had been successfully crystallized, and the complexity of members of this family had prevented evaluation through the genome sequence alone.

"This demonstration shows that cryo-EM is doable and is a major step in reaching the full potential of this technique," he said. "The goal is to have it reach a 3 to 4 angstrom resolution, which would allow us to clearly see the amino acids that make up a protein."

In electron microscopy, a beam of electrons takes the place of the light beam used in a conventional microscope. The use of electrons instead of light allows the microscope to "see" in much greater detail.

Cryo-EM cools specimens to temperatures well below the freezing point of water. This decreases damage from the electron beam and allows the specimens to be examined for a longer period of time. Longer exposure time allows for sharper, more detailed images.

Researchers using cryo-EM had obtained images at a resolution of 6-9 angstroms but could not differentiate between smaller elements of the structure spaced only 4.5 angstroms apart.

"There are different elements that make up the protein building blocks of the virus," Jiang said. "It is like examining a striped blanket. From a distance, the stripes blur together and the blanket appears to be one solid color. As you get closer you can see the different stripes, and if you use a magnifying glass you can see the strands of string that make up the material. The resolution needs to be smaller than the distance between the strands of thread in order to see two separate strands.

"By being able to zoom in, researchers were able to see components that blurred together at the earlier achieved resolution."

Cryo-EM requires high-end electron microscopes and powerful computing resources. The research team used the Baylor College of Medicine's cryoelectron microscope. It is expected that Purdue will install a state-of-the-art cryoelectron microscope in 2009.

In 2006 Purdue received a $2 million grant from the National Institute of Health to purchase the microscope. It will be installed in Hockmeyer Hall of Structural Biology, expected to open in 2009.

Computer programs are used to extract the signal from the microscope and to combine thousands of two-dimensional images into an accurate three-dimensional image that maps the structure of the virus. This requires use of a large data set and could not have been done without the resources of Purdue's Office of Information Technology, or ItaP, Jiang said.

Jiang used Purdue's Condor program - which links computers including desktop machines and large, powerful research computers - to create the largest distributed computing network at a university.

"ITaP provided us with computational power at the supercomputer scale that was necessary for this work," he said. "Purdue's Condor program allowed us to take advantage of the power of 7,000 computers. This was a critical element to our success."

Jiang plans to continue to refine every step of the process to improve the capabilities of the technique and to examine more medically relevant virus species.

Purdue's structural biology group studies a diverse group of problems, including cellular signaling pathways, RNA catalysis, bioremediation, molecular evolution, viral entry, viral replication and viral pathogenesis. Researchers use a combination of X-ray crystallography, electron cryomicroscopy, NMR spectroscopy, and advanced computational and modeling tools to study these problems.

Note for Capsid
A capsid is the protein shell of a virus. It consists of several oligomeric subunits made of protein. The capsid encloses the genetic material of the virus.
Capsids are broadly classified according to their structure. The majority of viruses have capsids with either helical or icosahedral structure. Some viruses, such as bacteriophages, have developed more complicated structures. The icosahedral shape, which has 20 equilateral triangular faces, approximates a sphere, while the helical shape is cylindrical. The capsid faces may consist of one or more proteins. For example, the foot-and-mouth disease virus capsid has faces consisting of three proteins named VP1-3.
Some viruses are enveloped, meaning that the capsid is coated with a lipid membrane known as the viral envelope. The envelope is acquired by the capsid from an intracellular membrane in the virus' host; some examples would include the inner nuclear membrane, the golgi membrane, or the cell's outer membrane.
Once the virus has infected the cell, it will start replicating itself, using the mechanisms of the infected host cell. During this process, new capsid subunits are synthesized according to the genetic material of the virus, using the protein biosynthesis mechanism of the cell. During the assembly process, a portal subunit is assembled at one vertex of the capsid. Through this portal, viral DNA or RNA is transported into the capsid. The structure and assembly of the Herpes virus Capsid Portal Protein has been imaged via cryo-electron microscopy.
Structural analyses of major capsid protein (MCP) architectures have been used to categorise viruses into families. For example, the bacteriophage PRD1, Paramecium bursaria Chlorella algal virus, and mammalian adenovirus have been placed in the same family.

Note for Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. The term is commonly used in its shortened form, phage.
Typically, bacteriophages consist of an outer protein hull enclosing genetic material. The genetic material can be ssRNA (single stranded RNA), dsRNA, ssDNA, or dsDNA between 5 and 500 kilo base pairs long with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy - usually between 20 and 200 nm in size.
Phages are estimated to be the most widely distributed and diverse entities in the biosphere. Phages are ubiquitous and can be found in all reservoirs populated by bacterial hosts, such as soil or the intestine of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 109 virions per milliliter have been found at the surface, and up to 70% of marine bacteria may be infected by phages. They are also found in drinking water and in some foods, including fermented vegetables and meats e.g. pickles, salami, where they serve the function of controlling any growth of bacteria.
They have been used for over 60 years as an alternative to antibiotics in the former Soviet Union and Eastern Europe, but have not yet been generally accepted elsewhere. In the case of MRSA, an infecting phage causes the bacteria to become more virulent and difficult to contain.
Bacteriophages may have a lytic cycle or a lysogenic cycle, but a few viruses are capable of carrying out both. With lytic phages such as the T4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the new bacteriophages viruses can find new hosts. Lytic phages are the kind suitable for phage therapy.
In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo lysogeny are known as temperate phages. Their viral genome will integrate with host DNA and replicate along with it fairly harmlessly, or may even become established as a plasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of nutrients, then the endogenous phages (known as prophages) become active. At this point they initiate the reproductive cycle resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is reproduced in all of the cell’s offspring.
Sometimes prophages may provide benefits to the host bacterium while they are dormant by adding new functions to the bacterial genome in a phenomenon called lysogenic conversion. A famous example is the conversion of a harmless strain of Vibrio cholerae by a phage into a highly virulent one, which causes cholera. This is why temperate phages are not suitable for phage therapy.

Note for NMR Spectroscopy
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is the name given to a technique which exploits the magnetic properties of certain nuclei. This phenomenon and its origins are detailed in a separate section on nuclear magnetic resonance. The most important applications for the organic chemist are proton NMR and carbon-13 NMR spectroscopy. In principle, NMR is applicable to any nucleus possessing spin.
Many types of information can be obtained from an NMR spectrum. Much like using infrared spectroscopy to identify functional groups, analysis of a 1D NMR spectrum provides information on the number and type of chemical entities in a molecule.
The impact of NMR spectroscopy on the natural sciences has been substantial. It can, among other things, be used to study mixtures of analytes, to understand dynamic effects such as change in temperature and reaction mechanisms, and is an invaluable tool in understanding protein and nucleic acid structure and function. It can be applied to a wide variety of samples, both in the solution and the solid state.
When placed in a magnetic field, NMR active nuclei (such as 1H or 13C) absorb at a frequency characteristic of the isotope. The resonant frequency, energy of the absorption and the intensity of the signal are proportional to the strength of the magnetic field. For example, in a 21 tesla magnetic field, protons resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, although different nuclei resonate at a different frequency at this field strength.
In the Earth's magnetic field the same nuclei resonate at audio frequencies. This effect is used in Earth's field NMR spectrometers and other instruments. Because these instruments are portable and inexpensive, they are often used for teaching and field work.
More subtle effects can occur if chemically equivalent spins (i.e. nuclei related by symmetry and so having the same NMR frequency) have different coupling relationships to external spins. Spins that are chemically equivalent but are not indistinguishable (based on their coupling relationships) are termed magnetically inequivalent. For example, the 4 H sites of 1,2-dichlorobenzene divide into two chemically equivalent pairs by symmetry, but an individual member of one of the pairs has different couplings to the spins making up the other pair. Magnetic inequivalence leads to highly complex spectra which can only be analyzed by computational modeling. Such effects are more common in NMR spectra of aromatic and other non-flexible systems, while rapid rotation about C-C bonds in flexible molecules ensures that the couplings between protons on adjacent carbons are identical, avoiding problems with magnetic inequivalence.

Note for Electron Cryomicroscopy
Electron cryomicroscopy is a form of electron microscopy (EM) where the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures). CryoEM is developing popularity in structural biology.
A version of electron cryomicroscopy is cryo-electron tomography (CET) where a 3D reconstruction of a sample is created from tilted 2D images, again at cryogenic temperatures (either liquid nitrogen or helium).
The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of the electron microscope column. Most biological specimens are extremely radiation sensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of cryo-electron microscopy provides an additional protective factor against radiation damage).
Consequently, the images are extremely noisy. For some biological systems it is possible to average images to increase the signal to noise ratio and retrieve high-resolution information about the specimen. This approach requires that the things being averaged are identical (e.g. ribosome particles). Analysis of ordered arrays of protein, such as 2-D crystals of transmembrane proteins or helical arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is called electron crystallography.
The thin film method is limited to thin specimens (typically < 500 nm) because the electrons cannot cross thicker samples without multiple scattering events. Thicker specimens can be vitrified by plunge freezing in ethane (up to tens of μm in thickness) or more commonly by high pressure freezing (up to hundreds of μm). They can then be cut in thin sections (40 to 200 nm thick) with a diamond knife in a cryoultramicrotome at temperatures lower than -135 °C (devitrification temperature). The sections are collected on an electron microscope grid and are imaged in the same manner as specimen vitrified in thin film. This technique is called cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-electron microscopy of frozen-hydrated sections.

Note for Electron Crystallography
Electron crystallography is a method to determine the arrangement of atoms in solids using an electron microscope. It can complement X-ray crystallography on proteins, such as membrane proteins, that cannot easily form the large 3-dimensional crystals required for that process. Structures are usually determined from either 2-dimensional crystals (sheets or helices), polyhedrons such as viral capsids, or dispersed individual proteins. Electrons can be used in these situations, whereas X-rays cannot, because electrons interact more strongly with atoms than X-rays do. Thus, X-rays will travel through a thin 2-dimensional crystal without diffracting significantly, whereas electrons can be used to form an image. Conversely, the strong interaction between electrons and proteins makes thick (e.g. 3-dimensional) crystals impervious to electrons, which only penetrate short distances.
One of the main difficulties in X-ray crystallography is determining phases in the diffraction pattern. Because no X-ray lens exists, X-rays cannot be used to form an image of the crystal being diffracted, and hence phase information is lost. Fortunately, electron microscopes contain electron lenses, and phase information tends to be much more reliable in electron crystallography.
A common problem to X-ray crystallography and electron crystallography is radiation damage, by which proteins are damaged as they are being imaged, limiting the resolution that can be obtained. This is especially troublesome in the setting of electron crystallography, where that radiation damage is focused on far fewer atoms. One technique used to limit radiation damage is electron cryomicroscopy, in which the samples undergo cryofixation and imaging takes place at liquid nitrogen or even liquid helium temperatures. Because of this problem, X-ray crystallography has been much more successful in determining the structure of proteins that are especially vulnerable to radiation damage.
The first electron crystallographic protein structure to achieve atomic resolution was bacteriorhodopsin, determined by Richard Henderson and coworkers at the Medical Research Council Laboratory of Molecular Biology in 1990. Since then, several other high-resolution structures have been determined by electron crystallography, including the light-harvesting complex, the nicotinic acetylcholine receptor, and the bacterial flagellum.

In figure 1, A colored electron micrograph of multiple bacteriophages

In figure 2, Icosahedral capsid of an Adenovirus

In figure 3, image of bacteriophage Epsilon15 studied by Wen Jiang, an assistant professor of biological sciences at Purdue. The bacteriophage is shown at a resolution of 4.5 angstrom - the highest resolution achieved for a living organism of this size.

In figure 4, Filtered CryoEM image of GroEL suspended in vitreous ice