BIOC 334: Exam 1

Important FSC cutoff values

0.143, 0.500, 3sigma or 5sigma. The smaller the FSC value the greater the resolution (more optimisitc value).

What is the minimum resolvable resolution by the electron microscope?

0.2 nm OR 2 Angstroms

How is resolution defined?

Estimated from the resulting reconstructed structure. We do the even-odd test and then generate two 3D FT and compare the correlation coefficient in shells of spatial frequency. This results in a FSC plot and where the plot drops below the threshold will be your resolution but converted to angstroms.

What are the EM grids?

Made of metal but coated in carbon. This is where samples are deposited onto. There are different types of grids: mesh, slot, and finder (used for reference numbers). It is best to use fresh grids because they are hydrophilic (spreading of aqueous beads) but they can be cleaned through glow-discharging or plasma-cleaning.

What do direct electron detectors do?

They can correct for electron beam-induced motion by having fast readout frames to measure and align the images.

What is inelastic and elastic scattering?

This occurs when the electron beam interacts with the specimen. When all energy is conserved, this is elastic scattering. When there is a change of energy, a secondary electron is generated which leads to a less focused image/blurry images.

How is ET resolution improve?

Through CTF or sub-tomogram averaging where we have multiple motor image sections from many tomograms.

How can CTF be altered?

Through changing the focus of the objective lens. Low defocus is when the high resolution phases contribute more and produces more detailed structures. High defocus is when low resolution contributes more and used to have more contrast. We can correction for image abberations by collecting images at different defocus values and then make computational corrections. We can also phase flip (change magnitude after experiment).

How are 3D images reconstructed?

Through combining multiple 2D projections at different angles. Then taking the FT of these (reciprocal space) and creating a 3D FT and then taking that reciporcal to find the 3D image in real space.

What is electron tomography used for?

To study individual protein molecules and complexes and also intact cells/tissues. It is used to study, unique cells. It is usually used to image large particles compared to small particles. The images are collected through tilt series, serial sectioning, and montage tomography. An incomplete 3D FT is produced due to missing tilt angles. (max tilt angle is 60-70 degrees). It also has a handedness (use of DNA origami gold nanoparticle with set chirality).

What is single particle analysis used for?

To study purified, identical protein molecules and complexes. High resolution is possible when orientation and conformation states (classes can be distinguished). Because we get imperfect raw images, we must attempt to correct for imperfections and limitations computationally and through averaging many images.

What is negative staining?

Use of solutions containing electron-rich heavy metal compounds to create a dark contrast between the background and samples. It is done at room temperature and typically done for preliminary studies. There is a greater contrast but lower resolution.

How are EM lens operated?

We can change the focal length by changing the current. We can also change the magnification of a single lens by changing the voltage and current. The purpose is to focus the electrons by bending and/or restricting their path. Lens are operated through solenoids.

How can single particle reconstruction be refined?

We can refine the relative orientation angles of the 2D projections using eulerian angles (projections based on relative angles to each other). We can also improve the cryoEM map through iterative proccess of comparing the images with projections.

Structure validation?

When covalent bond lengths/angles are close to ideal values so when deviats less than 0.02 A or 2 degrees. In staggered formation compared to eclipsed. We want side chain torsion angles to be in energetically favorable ranges.

How do we prepare for electron crystallography?

integral membrane proetins can be crystalized/purified through lipid layer crystalization. These 2D crystals then can be embedded in a sugar and dried (if done at room temperature) or can be plunge-frozen (if cryo-diffraction). We then can measure many images of the crysal and diffraction images at different tile angles.

What is DD?

Direct detectors don't have an intermediate layer so there is reduced scattering and noise. There however is required more storage capacity.

What is CTF?

Contrast transfer function is calculated to describe the aberrations in teh raw image and then is used to correct for these mistakes. It is calculated from a rotationally averaged power spectrum (FT transformation of the raw image). The radial cross-section is plotted against spatial frequency and there are bands of resolution that contribute more to the image.

What is CLEM?

Correlated light and electron microscopy is used in ET to chemically tage an object and then image by fluroscent microscopy.

What is the procedure for single particle reconstruction?

1. Image collection at different orientations/angles. 2. identify particle images. 3. sort them into different classes based on different orientations and conformations. 4. Average the classes to improve signal:noise which results in higher resolution and contrast. 5. Take FT and produce 3D image. This can be iterative.

What are the challenges in EM?

1. Preserving native structure within the chamber of the microscope since the macromolecules are in hydrated environment. This is why sample preparation is so important. 2. Obtaining 3D structural information from 2D projections. 3. Dose limitations due to potential radiation damage from the high energy electrons.

How does one limit radiation damage?

Because intensities of the diffraction spots will decrease as radiation damage increases, we need to limit radiation damage. This can be done by cooling; however, should avoid super cooling. We can also measure many images at low doses and average to increase the signal:noise.

How are electrons generated?

By heating or applying a voltage to a cathode and then applying an accelerating voltage for the electrons to go towards the anode. The greater the accelerating voltage, the smaller the wavelength thus the higher frequency (energy) of the electron beam. Typical electron source include Tungsten filament, Lanthanum hexaboride, and Field Emission Source.

What is CCD?

Charged-coupled device has extra scattering of electrons within the scintillator and fiber optic layers which causes limited resolution and sharpness. There is also extra noise.

What does a condenser lens do? What does the objective lens do?

Focus the electrons on the specimen. Focus the electrons that have been scattered by the specimen.

Challenges of electron crystallography?

Hard for crysals to be flat, hard to find 2D, images can be blurred due to beam-induced movement, there are tilt angle limits. We can do helical tubes (have molecules in a large number of orientations in a single image which leads to equivalent of a complete range of tilt angles).

What is electron diffraction used for?

If 2D crystals available, can yield high resolution structures. Because some crystals have ordered molecules aligned in a pattern that can produce measurable diffraction through the unit cell. We need to know both the amplitude and phase of the diffracted waves for FT calculations. Electron diffraction uses 2D crystals and then does a FT to get reciprocal difraction pattern. Ampltiudes are from diffraction pattern while phases come from magnified image.

What does a lens do in a microscope?

It focuses the diffracted waves to bring them into constructive interference in order to reconstruct an image of the object.

How are objects in a tomograph identified?

Obvious features, CLEM, by matching via EM, perturbing abundance/structure, template matching with CCF (cross correlation function) where we compare the different angles of the templates in a parallel computation.

What is cryoEM?

Plunge the grid into liquid ethane and then into dry ice (nitrogen) in order to image. There is a greater resolution but a lower contrast. It is used for more detailed structures. This is done because there is a rapid freezing of the thin sample layer before the ice crystals can form.

What is scanning EM used for? What are the other transmissive EM?

Scanning EM is reflective and it is only used to image the surface. Transmission EM and Scanning tunneling EM.

How are lens imperfect?

Spherical aberration (further an electron is from the axis, the more distorted the image), chromatic aberration (different energies of electrons are not shown as the same color), Astigmatism (non-uniform magnetic field)

Why would one use electron microscopy over light microscopy?

The electrons have a smaller wavelength which allows for a greater resolution. Because of their wavelength, it can also be focused better.