Determinazione di strutture molecolari di glicoproteine ​​dell'HIV Busta con Cryo-Electron Positron e automatizzato Sub-tomogramma Averaging

Over 60 million individuals have been infected with HIV, the virus that causes aids. HIV AIDS is a global health crisis, and although intensive research efforts have dramatically lend them the lives of infected individuals, a true vaccine remains elusive. A major focus of HIV vaccine research is the envelope glycoprotein, which HIV uses to enter target cells.

In this video, we demonstrate how we obtain structures of this important protein using an imaging technique called cryo-electron tomography. Beginning with purified HIV, we show steps in preparation of frozen specimens, steps in recording electron microscopic images from these frozen specimens and steps in converting these images into structural models that could be useful for hiv aids vaccine design. A unique aspect of what we present in this video is that the work will be demonstrated by members of her laboratory spanning a wide range of ages from middle and high school students to college and graduate level students all the way to postdoctoral fellows and senior scientists.

The preparation of vitrified virus samples suitable for cryo-electron tomography is accomplished by spreading an aqueous suspension of viruses across small holes in a carbon film supported by a copper grid, and then rapidly freezing the viruses. The goal of this step is to prepare a frozen specimen of intact viruses captured in a near native state. To begin, the grids are placed inside a glow discharge unit.

This device cleans the grids by exposing them to ionized gas. Now that the grids are ready for use, a suspension of viruses mixed with small gold particles is prepared. These particles are essential for data collection and processing.

Next, a drop of virus suspension is placed on the freshly plasma cleaned grid. The grid is then loaded in a robot, which blots it with filter paper, reducing the drop to a thin film of liquid. The sample is then plunged into liquid ethane, which rapidly freezes the viruses at a rate exceeding 100, 000 kelvin per second.

The plunge frozen grid is then transferred to a storage box for transport to the microscope. This is repeated until the desired number of grids are frozen. Now that the virus sample grids are ready, the next step is to load them into the microscope.

For imaging, this can be done manually or with robotic assistance. When the sample grids are loaded manually, the user sits at a specialized station where the samples can be manipulated under liquid nitrogen, the samples are placed into a portable nitrogen cooled chamber that is sealed, evacuated, and then attached to the microscope. Newer microscopes are capable of more automated sample loading.

Here in a Titan Cryos microscope, frozen samples are delivered to the microscope by the user and then loaded robotically with the sample grids loaded in the microscope. Each grid can be examined by the user before imaging can begin. The microscope parameters are set up for each area on the sample grid that has features of interest.

In this case, the features are HIV viruses. As each new area is defined, the microscope computer stores the area specific imaging parameters. When the user has defined all positions of interest, the computer revisits each position automatically and collects a data set.

Each dataset is collected by tilting the sample grid with respect to the electron beam, while ensuring that the beam stays focused at the same spot on the grid. Data collection in electron microscopy has traditionally required users to sit at the microscope and physically interact with the equipment. This style of microscope interaction is shown here during a visit from President Obama to an intel factory.

Recent advances in microscope computer interfaces now allow users to monitor and adjust data collection in real time from a remote computer at the laboratory or elsewhere. In order to visualize the shapes of the envelope, glycoprotein spikes the information from hundreds of individual images must be averaged to boost the signal. This is a computationally intensive process, which is accomplished here by using a cluster of computers called Bio Wolf.

Located at the NIH, powerful methods have been developed to extract individual volumes and average the information using algorithms that can work at the high noise levels. Intrinsic to this type of data, the density maps obtained from both native viruses and viruses complex to biologically important molecules provide a rich database of information on the structural biology of HIV. The density maps come alive at molecular detail when the data is combined with information from x-ray crystallography of the individual subunits that make up the envelope glycoprotein spike.

Data that comes out of these tomos can be very dense and challenging to interpret. 3D software for the entertainment industry can be used to visualize the 3D models by adding colors, textures, and lighting, making them easier to analyze. Commonly used visualization software includes a Mirror 3D Studio Max and Maya.

We have just demonstrated step-by-step procedures for how one can obtain molecular models for the structure of the envelope. Glycoprotein, starting from purified HIV. Using this method, we have also determined structures for the envelope glycoprotein in complex with a variety of molecules that can bind and utilize the virus.

Here we show an example for the structure of the envelope glycoprotein in complex with an antibody called B12. Understanding why some molecules can bind and utilize the virus while others do not is very important for rational vaccine design. Determining molecular structures of envelope glycoproteins from HIV or from other viruses, such as influenza and Ebola, is key to understanding viral entry and utilization.

As you have seen, cryo-electron tomography is now emerging as a powerful tool to achieve this goal.