Lecture 5 Flashcards
What is EM limited by ?
Sample preparation. The resolution of EM is limited by the resin we use
Max resolution for EM?
50 angstrom
Two ways to retrieve 3D images from 2D?
- Serial sections
- Tomography
Serial sections
- Cut the sample using the ultramicrotome and keep the sections in order(trapezoid)
- Image each section in the EM
- Stack of the sections to get the full 3D image
How is serial sectioning different from classical EM?
Section the sections thinner
Problem with serial sections
Resolution is Z is limited by the thickness of the section can only go up to about 30 nm
What is tomography?
-Cut a thick section and put it in the EM and take pictures of it from various angles. We know the angle from which we took the image so then we construct the 3D image via back projection
CT Scan
Human sized version of tomography
Tilt series
When we collect the stack of images from various angles
Problem with tomography?
-Can only deal with 300nm sections
-Cannot deal with a cell (too big)
How do we deal with cells for tomography?
We do serial section tomography
What is serial section tomography?
-Cut thick sections of various sections of the cell
Then do tomography on each section
Then stack all of the tomography sections together to get the full 3D image
Problems with Fixation?
-Slow (glutaraldehyde is slow)
-Conformation changes of protein
-Permeability change of membreane
-Osmotic effect leads to dimensional alteration
Problems with dehydration?
-Shrinkage
-Conformational change of proteins
-Loss of lipids
Porblems with embedding?
-Shirnkage during polymerization
-Loss of lipids
Problems with thin sectioning?
-Compression
-Knife marks
Problems with staining?
-Artifacts
Problems with TEM?
Interpretation mistakes
How to overcome dehydration altering what we look at?
-Cryo-EM
What is Cryo-EM?
-Frozen hydrated state
Vitrification?
transformation of a substance into a solid state that is a non-crystalline amorphous solid
How does Cryo-EM do vitrification?
By plunging a small volume of sample quickly into liquid Ethane at liquid nitrogen temperature
-Freezes the sample so fast that crystals don’t form
Why does Cryo-EM imporve ultrastructure preservation?
-Instead of using fix we just freeze the sample which is faster
-No ice crystals form which would damage the sample
-Forzen hydrated( solid but in a liquid state(still hydrated))
Leidenfrost Effect
The formation of a gas barrier between a hot surface and a boiling liquid if the temperature is great enough
Why can’t we vitrify the water in the sample using liquid nitrogen?
Temperature difference between the water in our sample and liquid nitrofen is so big the gas barrier will form and cause our sample to freeze slow which creates crystals
Cryo-electron tomography?
- Vitrify your sample
- Put the sample in the EM and take tomographic pictures
- Back reconstruction to get the 3D image
How is cryo EM better than resin?
-Better ultrastructure preservation
-Best resolution
-Low contrast
Why does cryo-EM have low contrast?
-Limit amount of electrons we can use on the sample since it is still hydrated
What are the benefits of visualizing atomic details in Cryo-EM?
Understansing things at the molecular level(how ribosomes do translation)
What technique allows us to visualize atomic details?
Single particle cryo-EM
Requirement for single particle EM?
Must use purified protein complexes not organelles or cells
Particle Picking?
Choosing the particles in the cell that are at varying orientations
2D classification
Grouping of the ribosomes based on their appearance, same orientation, same conformation or same composition
3D alignment & reconstruction
- Match our expierimental image with the 3D projection(whichever one matches best)
- Then do tomography and do backreconstruction to get the 3D structure
Cryo-EM for structure determination advantages?
-Easy sample prep
-Molecules close to native state
-Requires only a small amount of sample
-More forgiving on sample purity
Jacques Dubocht
Vitrification
Joachim Frank
Computational of the 3D structures
Richard Henderson
Instrumentation and Imaging
