Electron Microscopy 2 Flashcards
Can em teach us more
Most current knowledge = based on 2d images - classical tem images are 2d projections
Cells and tissues are 3d - can we get 3d info in tem
Is everything observed to date real - resin embedding = harsh ans causes deformation - can we do better
Describe our current knowledge of molecules
50 angstroms - in resin - resolution limited buys ample preparation not tem
Can em get better resolution - through sample prep and methods of image analysis
Why is 2d not enough
2d projection can give v misleading info about 3d structure
How to retrieve 3d from 2d
Serial sections
Tomography
Describe serial sectioning
Er- complex morphology = need 3d image
Imaging of consecutive sections from ultramicrotomy for 3d view - each separate, keep every section in order = stack and get 3d
Serial section from sectioning allows 3d view of structure - resolution limited by section thickness
In z= depends on cutting, best = 30nm but v high
How are serial sections done
Stack manually or automatically using tool
Need confocal lm - sections thinner, dept of field small = better resolution
New Vision of the er
Using novel ultra thin sectioning technique - in which sections are 30-40nm
Stacked er sheets connected by helicoidal membrane motifs
What is tomography
Like ct scan
Resolution of serial sections limited bu thickness of sections - 50-100nm - thinner sections lead to better resolution
Tomography allows to get a better 3d info about structures in thickness of section -300nm
Describe principle of tomography
Take pics from diff angles
3d object —> set of 2d projections —> 3d reconstruction
Know angle = then do back projection - based on degrees of pics and can reconstruct
-60-100degrres = 61 images then back project to 3d structure
Describe tomography history
3D reconstructions from projection images:
– Cormack and Hounsfield: Nobel prize in 1979 for Xray computed tomography
3D reconstructions from
EM images:
- Klug: nobel prize in 1982 for applying tomography principle in TEM (DeRosier and Klug 1968)
Ct scna invented, computer happened - calculate and back project images
Tomography waasnt poplar until 2000s —> bc need to rotate em by hand = hard but now automatic and computer controlled
Describe tilt series
Data collection = stack images from diff angles
= can get reconstructed tomography lots of detail, only 300nm section could do 5 micron cell —> serial section and tomography but hard
Problems with conventional em sample prep - fixation
Slow= seconds to mins
Conformation changes of protein
Permeability changes of membranes
Osmotic effect leads to dimensional alteration
Loss of diffusibel ions and small molecules
Masking of antigens
Problems with conventional em sample prep - Dehydration
Shrinkage
Conformation changes of proteins
Loss of lipid
Problems with conventional em sample prep - Embedding
Mechanical effects = hardening
Shrinkage during polymerization
Loss of lipids
Problems with conventional em sample prep - Thin sectioning
Compression
Knife marks - if not perfectly sharp
Problems with conventional em sample prep - Staining
Staining artefacts - precipitation of heavy metals
Problems with conventional em sample prep - Tem
Interpretation mistakes
Is ervything observed to date real
Biological sample is 70% water. Classical processing: Totally remove H2O and we cook it!!! Does that really preserve the structure of cellular component?
Technological adavances = can look at samples in more native conditions
= cryo em - observation of sample frozen
Describe what happens when biological sample frozen
Cannot just freeze or use liquid nitrogen = damage = forms crystallized ice
Describe vitrification
Transformation of substance into a glass — a non crystalline amorphous solid
Sample to be in solid state but non crystallized form
Liquid state but behaves as solid
Describe cryo em
Vitrification of biological samples in solution on em grid by plunging small vol of sample quickly into liquid ethane at liquid nitrogen temp
= freeze so quick no crystals formed
Suspension sprayed = tiny droplets —> through slit —> on to em grid - has lots of particles —> dip in liquid ethane
Why do cryo-EM improve ultrastructure preservation?
Instead of using chemicals to fix = freeze v fast = so everything in place
Ice crystals damaging
If freeze v fast - at least 10,000 degrees Celsius/second = ice crystals do not form but amorphous ice = vitrified water
Observe sample at cold temp = no ice crystals
Sample frozen hydrated and in its native state
Why liquid Ethane and not liquid nitrogen for cryo em
Liquid ethane = -72degrees
Liquid nitrogen = -195 degrees
Water at room temp = 20degrees
If use liquid nitrogen = leidenforst efefct = will happen and will crystallize
Leidenfrost effect = formation of gas barrier between hot surface and a boiling liquid if temp diff great enough
Compare cryo em to classical em
See more detail but distorted with classical
See evrurthing with cryo (could do sectioning with cryo em but super hard = have to cut vitrified ice, electrostatic so section sticks on knife)
How to see proteins directly in cell in cryo em
Thin area or cellular organelles
Cryo electron tomography
What does cryo em give us - image
3d tomography
Can see thin structures
But low contrast images compared to resin
Structure biological sample and must limit e- exposure = radiation of sample
Can we see atomic details in cryo em
Higher res
Need to understand how cells functions at molecular level and how proteins function
= use single particle em
What do we see at high resolution in tem
Cannot see well in regular classical em = need high magnification or higher res images
Describe single particle analysis - gen
Applies to protein complexes
Assign orientation of projections in relative to 3d structure and calculate the reconstructed 3d structure
Describe acquire protection images of single particle analysis
Purified proteins vilify and put on grid and take pics = Acquire projection images
Of diff ribosomes at diff orientations
Describe classifying projections of single particle analysis
For similarities = then see that there are similar groups = can improve signal to noise = have 2 diff classes =
Assume ribosomes have same structures = imaging single particle at diff orientations —> figure out orientation of projections and then do back projection = get 3d structure of ribosome with single particle assumption
What is requirement for single particle analysis
Purified protein complexes not organelles or cells
Describe cryo em workflow
1- purify = sample prep
2= vitrify then sample freezing and loading
3- automated image acquisition
4= data storage and handling
5= to get image processing = 3d reconstruction
Describe single particle analysis particle picking
Identify location of protein complexes - particles in the image
Manual picking = used to be manual
Vs
Automatic picking = software
Describe single particle analysis classification
2d images need to be grouped based on their appearance - same orientation, same conformation and same composition
Used to discard wrongly picked particles or damaged particles
Then average them to improve signal to noise ratio
Describe single particle analysis 3d alignment and reconstruction
Projection of initial model = protein we looking at low res —>
Model 3d projection, matching of experimental image with projection —>
Reconstruction = assign kno angles of proteins = we have it to image =
3d structures = high res
Describe cryo em fro structure determination advantages
Easy sample prep
Molecules in closer to native state
Requires only small amount of sample - 0.1mg
More forgiven on sample purity
Give info on sample dynamics
Structure of 2019 cov spike in perfusion conformation
Spike glycoprotein = in receptor of cilia, can understand life cycle -> can find Therapy for it
Ex of single partical cryo em
Plasmid that expresses spike protein
Vitrification and do single particle cryo em
3d variability analysis = see binding domains, first structures of spike proteins = help design vaccine
Covid variant = location of mutation in spike proteins = can see this
What makes omicron so transmissible = put in receptor too, mutated = weakened antibody binding but still strongly binds receptors
What can we see using singe particle cryo em
Full structure of ribosome
Microtubules very clearly
Actin filaments
Tau proteins
What allows for Best preservation of samples
Very rapid freezing - cryo em
What happens when frozen hydrated samples are hit with electrons
Frozen hydrated samples v sensitive to electron beam
Amount of electrons required to get 3d high resolution info from a frozen section will destroy sections much faster than images can be collected
How to see purified protein complexes
Using averaging methods or single particles population average = average many images of identical complexes = purified protein complexes
Do we stain with cryo em
Nawww
Only contrast = density difference between proteins and water = v low
Defocus to get more contrast then reform image (bc deforms) = more contrast - can also use special cameras
Describe single particle vs tomography
With spa = have to separate things
Cannot overlap particles, cannot use for almost everything inside cells
What destroys high res info
Resin embedding
