Doryen Bubeck - Cryo-EM Flashcards
Outline the importance of understanding the fourier transform for Cryo-EM? Why are we learning about fourier transforms in the context of Cryo-EM?
Outline the three key parameters of a wave?
Remember
Frequency = 1/wavelength
How can the following wave be plotted in Fourier space/Frequency space?
Note - There are two plots one against Amplitude and the other against Phase!
Application of the fourier transform to the ear/brain perceives sound?
In it’s essence what does a Fourier transform do?
In its essence what does a inverse Fourier transform do?
What’s it the Fourier transform of this wave? What are the consituent waves?
How can we represent 2D waves as in 2D space & grey scale oscillations?
What is the wavelength and frequency for both plots?
What does a Fourier Transform of the attached 2D wave look like?
What does a Fourier Transform of the attached 2D wave look like?
What does a Fourier Transform of the attached 2D wave look like?
What happens when we combine two multiple 2D sine waves?
Summary of the Fourier Transform?
- Waves can exist in 1-D, 2-D, 3-D described with an amplitude, phase, and 1-D, 2-D, 3-D frequency component(s).
- The Fourier transform of a wave is a way to represent a wave in terms of its frequency instead of wavelength: i.e., in “reciprocal space or frequency space”.
- Multiple simple waves can be combined to form a composite wave –> these component sine waves of composite waves can be discerned in reciprocal space
How can we apply the ideas of Fourier transform of waves to actual images?
What happening here?
How will changing a single pixel in the image impact the Fourier transform
If you change a single pixel in the image - it will subtlety change every Fourier component
This is information is delocalized
Just like in X-Ray crystallography - every dot on the detector has information for the entire molecule
What impact would removing Low spatial frequencies and high spatial frequencies have on our image?
What is important phases or amplitdues when it comes to producing an image?
Sublecture 2 - summary?
What are Friedel pairs? Why do they appear?
Does every Fourier transform have a DC component?
Difference between X-Ray and Cryo-EM when it comes to Fourier and Inverse Fourier transform?
What’s all the fuss about? Why is Cryo-EM important?
What can we image with Cryo-EM?
Why is Electron microscopy an amazing idea from a physicists perspective?
Why is Electron microscopy not so simple from a biologists perspective?
Who were the Nobel Prize Winners in 2017 for Cryo-EM?
- Jacques Dubochet
- Joachim Frank
- Richard Henderson
What was Dubochet’s contribution to the 2017 Nobel Prize?
What was Frank’s contribution to the 2017 Nobel Prize?
What was Hendersons contribution to the 2017 Nobel Prize?
Is the quality resolution of Cryo-EM structures increasing over time?
What does the resolution revolution refer to?
Differences and similarities between Electron and light microscope?
Why do we need electrons to image biological structures?
What are the two ways in which a electron can behave?
When an electron encounters an atom what are the different possible interactions?
What are the different componenets of an electron microscope?
What are the two possible sources of electrons?
How are electron waves focused?
What are the three main lenses in the electron microscope? What are their respective functions?
What is the function of the apertures in the EM?
How are images detected on a electron microscope?
Why are Direct electron dectectors particularly useful for cryo-EM?
Increased sensitivity allows us to expose our biological samples to lower levels of electrons - reduces the amount of ionizing radiation/reduced damage + allow for movie feature
Why is the movie feature (obtaining snapshots) important for cryo-EM?
What does this image show?
With refrence to Cryo-EM, what does contrast mean?
In Cryo-EM, what are the different ways in whic amplitude contrast is generated?
In Cryo-EM how is phase contrast generated?
Outline the progression of waves as they are focussed by the objective lens?
What would the vector diagram for the following scattered and unscattered wave look like?
Example of negative contrast - Sum of the waves is less than the unscattered waves
What would the vector diagram for the following scattered and unscattered wave look like?
Example of 0 contrast - Sum = unscattered
What would the vector diagram for the following scattered and unscattered wave look like?
Example of positive contrast - Sum > unscattered
What is one important consequence that arises from the different scattering angle of scattered waves?
Hint - oscillations
What is one effect of the lens that we can not control?
Is defocusing an important part of collecting images with sufficient contrast?
Using the Contrast Transfer function (CTF) explain why it is important to use different focus/defocus ranges?
Defocus ranges - refers to changing the strength of the lens resulting is more or less bending
What are the effects of the CTF on an image?
Delocalizes the signal & inverts the contrast at different resolution ranges (important correction – without it we get distortions) –> need to bring everything back up to +-ive contrast
Apart from the objective lens, what is something that can be employed to generate contrast?
When preparing proteins for Cryo-EM, what are three key things we need to consider?
Is obtaining pure protein samples important for Cryo-EM
Apart from protein purity what other types of heterogeneity do we need to consider in Cryo-EM?
What is the overarching goal of sample preparation?
What are the three main problems we need to consider when trying to acheive high resolution images?
What is negative stain EM? Why is it used?
Outline the procedure to obtain negative stain images?
In Negative Stain EM, how is the sample loaded into the EM?
Advantages and Disadvantages of using negative stain EM?
Outline how our sample is vitrified?
How does the Double bath system for vitrification work?
Double bath system
- Shows liquid nitrogen bath that is cooling the whole system
- Ethane bath in the middle that is actually used to freeze the sample as it gets plunged in
Is all the Ice created by the vitrification procedure the same?
What would be the ideal scenario when performing vitrification?
What is one of the main difficulties that arises when freezing our sample?
How can we minimize the impact of the air-water interface?
After freezing but before loading our sample on to the microscope, where are our grids stored?
Dewar*
In Cryo-EM, does exposure time correlate with radiation damage?
What is one way that we minimize radiation of our sample?
Hint - Focus and Underfocus
Low Dose Sample Navigation
One we have decided where we want to collect our images? Which Hole?
We find Focus and under focus areas to create contrast using objective lens but importantely…
Find focus away from where you are going to expose your sample - prevent pre-irradiation
What this means is that we are going to shoot blind!
Apart from shooting blind, what else can done to minimze radiation damage?
Is radiation damage inevitable?
Briefly outine the Cryo-EM process.
What is the relationship between 2D images & 3D objects?
What is the goal of Cryo-EM single-particle analysis?
Recap - what does the Fourier Transform allow us to do?
What is the projection theorem?
How can we apply the projection thereom to EM?
How do we determine the orientations of the 2D projections? Why is accurately determining the orientation important?
What impact does averaging over single-particles have on our image?
Outline what is going on in this image.
Is beam induced motion correction important?
During image processing what does defocus estimation refer to?
Is CTF correction important - image processing?
Once Defocus estimation and CTF correction have taken place, what do we need to do?
What does Particle alignment and classification refer to?
Particle alignment and classification
Then we try to match particles that are similar and average them to classify them into the same group - taking particles with the same view and orientation
Describe what happening in the attached image.
In Particle alignment and classification - what does Projection matching refer to?
Outline the reconstructing the image procedure?
What are different techniques that can be used to minimize the possible negative impact of conformational heterogeneity?
How do you whether your Cryo-EM model is correct?
What does Model Bias refer to? How can we minimize it’s effects?
What are some potential improvements to consider during sample prep to facilitate imaging?
- Minimizing loss of sample when blotting
- Changing the support to reduce beam induced motion (Ice tension) and minimize impact of water-air interface
What properties of a sample make it ideal for Cryo-EM?
- Proteins located in a lipid bilayer (Membrane proteins) – no need to solubilize proteins in detergent allowing for more native-like environment
- Cryo-EM more suitable for larger proteins
- Limited supply of protein is needed - beneficial if it is hard to purify
Role of negative stain in the imaging procedure?
Is it okay for your sample to be a little bit heterogenous?
What is the Air-Water interface?
What factors influence how your particles are orientated in the Ice?
Can negative stain be used to discern orientations of 2D projections?
Yes its is possible!
But abInitio methods done computationally are done more frequently
Outline the Finding Focus and Defocus procedure?
How do we apply focusing/defocusing procedure when performing low dose imaging?
We find focus/defocus around the region we want to image - cause we want to limit radiation damage
This means we are shooting the exposure area blind.
