What is single particle reconstruction?

Single Particle Reconstruction is a method used in 3D EM when multiple copies of. the particle of interest are present. We expect that the particles are present in different orientations on a EM grid (to. have all projections, angular coverage)

What is single particle electron microscopy?

The phrase single-particle cryo-electron microscopy (cryo-EM) refers to applications of electron microscopy (EM) in which specimens consist of randomly dispersed, unstained biological macromolecules. Such specimens are embedded in a thin film of vitrified buffer, thus preserving their structure in a near-native state.

What factors cause image distortions in cryo-EM?

In cryo-EM, two factors lead to image distortion—electron beam-induced local motion of the specimen and defocusing of the electron lenses required for increasing the phase contrast.

What is cryo-EM reconstruction?

Single-particle cryo-electron microscopy (cryo-EM) is a powerful method for determining the atomic structure of macro-molecules by imaging them with electron rays at cryogenic temperatures [1–3]. The reconstruction procedure in single-particle cryo-EM remains nonetheless riddled with complex obstacles.

What is single particle imaging?

Single particle analysis segments and averages many particles from a sample, allowing for computer algorithms to process the individual images into a combined “representative” image.

How does electron tomography work?

Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data. In the process, a beam of electrons is passed through the sample at incremental degrees of rotation around the center of the target sample.

What is b factor in cryo-EM?

In cryo-EM, the most widely applied method is a structure factor modification based on the Guinier plot, also known as B-factor correction (Rosenthal and Henderson, 2003). Thus, global sharpening determines a B-factor value, which is then applied across the whole map.

What is defocus in tem?

In TEM image observation, the focus of the objective lens is shifted for observing Fresnel fringes or for taking a lattice image or a structure image. This focus shift is called “defocus.”

How much does a cryo-EM cost?

Henderson, Russo, and a small group of confederates are trying to make cryo-EM affordable. A top machine costs about $7 million. Preparing a room and installing a microscope can cost just as much. Then come the operational costs—a torrent of electricity, dedicated troubleshooting staff—that can rise to $10,000 per day.

Why is cryo-EM?

Cryo-EM does not require large sample sizes or crystallization and is therefore suited to the visualization of structures at near-atomic resolution. The method also has the advantage of not chemically fixing or staining the specimen, meaning it can be studied within the native physiological environment.

What is a disadvantage of electron microscopes?

Electron Microscope Disadvantages The main disadvantages are cost, size, maintenance, researcher training and image artifacts resulting from specimen preparation. This type of microscope is a large, cumbersome, expensive piece of equipment, extremely sensitive to vibration and external magnetic fields.

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