Cryo EM Flashcards

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1
Q

Advantages of cryo EM

A

• Allows molecules to be studied in near native environment
• Biochemically functional buffers
• Can look at membrane environment
• Functionally relevant conformations
• No crystal packing artefacts
• X ray crystallography can trap non relevant conformations as it needs to pack that way to make the crystal
• Relies on only a few micro litres of material
• Concs as low as 10s of nanomolar
• Can image large scale of complexes, including macromolecular assemblies
• Can get atomic distances
• Cells, bacteria, viruses, proteins, atoms

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2
Q

Why is EM the resolution revolution

A

• High energy e- are waves
• Wavelength is smaller than inter atomic distances
• Resolution is proportional to wavelength
• Diffraction limit for EM is not a problem like for light microscopy
• These e- can be focused by magnetic lenses
• Images more powerful than diffraction patterns (images contain amplitude and phase info)
• Don’t need to solve phase problem

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3
Q

Issues with EM from biologists perspective

A

• Em operate at high vacuum to avoid unwanted scattering of electrons – means all the water would be sucked out of sample
• High energy e- are not just short wavelength waves but also ionising radiation – means you destroy sample when you image it
• Forced to limit exposure of sample to the electron beam to avoid damage – means you collect noisy images
• CryoEM samples move when irradiated (beam induced motion) – blurry pictures

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4
Q

Dubochets vitrification method

A

• Sample prep method
• Sample transferred to metal mesh and excess material removed
• The sample forms a thin film across the holes in the mesh when its shot into ethane at about -190 deg C
• The water vitrifies around the sample, which is then cooled by liquid nitrogen during the measurements in the electron microscope
• Allows sample to be looked at in vacuum condition

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5
Q

Frank’s image analysis for 3D structures

A

• Randomly oriented proteins are hit by the e- beam, leaving a trace on the image
• The computer discriminates between the traces and the fuzzy background, placing similar ones in the same place
• Uses Fourier transforms
• Using thousands of similar traces, the computer generates a high-resolution 2d image
• The computer calculates how the different 2D images relate to each other and generates a high resolution structure in 3d

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6
Q

Henderson’s vision for high resolution cryo em

A

• Worked on 2d crystals of membrane proteins
• Pioneered low dose imaging
• Was aware of what damage the radiation was doing to samples
• Pioneer of direct electron detection for cryoEM
• Increased sensitivity of the image and reduced radiation dose

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7
Q

What is the Rayleigh criteria

A

Defines the theoretical resolution limit
Resolution is the ability to resolve 2 points
Distance between 2 points = (wavelength of radiation x 0.61) / numerical aperture of the lens
• Visible light has a wavelength of 400nm so would have theoretical resolution of 200nm
• E- have wavelength of 0.0025nm so have theoretical res of 0.0015nm
• E- display wave particle duality

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8
Q

Compare the types of interactions electrons can have with a sample

A

• Most (80%) e- pass straight through sample- transmitted
• Some are elastically scattered by the nucleus and give the signal for the image
• Some are inelastically scattered and release energy in the form of ionising radiation, this contributes to noise level and radiation damage
• Some e- are back scattered so don’t contribute any info to the image
• The fundamental challenge is balancing signal and noise

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9
Q

Major elements of an electron microscope

A

• Electrons are emitted from a source
• E- generated at top of microscope
• Can be from tungsten filament
• Accelerate e- and heat up the filament
• E- are extracted from the pointed tip under a vacuum
• Field emission guns are also used as they have a much better coherence of the e- beam than tungsten
• Lens is made up of a series of copper coils
• E- wave passes through the centre
• Focus the e- by applying different currents through the copper wire

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10
Q

3 lenses of the EM

A

• Condenser lens: lenses control intensity, brightness, coherence and convergence of e-
• Objective lens: important for generating contrast
• Projector lens: amplification, magnifies image before detector

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11
Q

Apertures of the EM

A

• Apertures remove highly scattered e-
• Blocks highly scattered e- from going further down the column
• 1 set after the condenser lens to reduce spherical adoration
• 1 after objective lens to increase amplitude contrast

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12
Q

How is EM image detected

A

• Used to use film camera
• Used CCD camera but had bad resolution
• Can now use DD sensor to collect quick frames and compile into a video
• ID sensors collected an e- signal which was converted to a light signal then back to an e- signal so there was a loss of info in the process
• DD sensors have no info loss as they detect signal from the scattering an a single e-, increase the sensitivity so you can detect with less dose and can make movies with short frames

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13
Q

Why do particles move in the ice and how do we fix this

A

• Freezing the sample changes the potential energy of the system
• The e- release some of the tension from the sample so some of the particles can move
• By aligning frames of the movie you can correct rotation and get a clearer image

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14
Q

What are the 2 types of contrast

A

• Contrast is the difference in intensity between scattered and unscattered waves
• 2 types:
• Amplitude contrast (due to particle properties of e-)
• Phase contrast (due to wave properties), very important carrier of info

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15
Q

What is amplitude contrast

A

• Due to differences in thickness(not super important as sample is very thin), density (important in -ve staining microscopy) and lattice (for e- crystallography of 2D crystals)
• Enhanced by objective aperture as highly scattered e- contribute to noise

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16
Q

What is phase contrast

A

• E- move down the column of the microscope as a planar wave from the source
• Encounter atoms of a sample
• Causes phase shift of the planar waves
• When they are scattered they become complex waves which are a sum of planar waves and spherical waves

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17
Q

How do you focus with the objective lens

A

Incident wave is planar
• Interacts with scattering centres
• Resultant is an unscattered planar wave ( from the transmitted e-) and a complex wave
• Scattered wave is scattered 90 deg phase shift
• Lens causes an additional phase shift to focus the wave, this changes the amplitude of the wave
• Many waves are being scattered
• Higher scattering angle = higher resolution
• Waves superimpose to create a resultant wave

18
Q

What is the effect of the lens

A

• Electrons are bent more strongly on the periphery
• Lens causes additional phase shift
• Strength of the lens is not uniform across the whole length – stronger at higher scattering angles
• Images that are in focus have 0 contrast
• Defocus = more contrast
• You want to under focus your image to get contrast
• Contrast is the difference between the unscattered and scattered waves
Too much defocus will give artefacts

19
Q

What happens when you change the focus

A

• Changing the focus changes the wave function
• Oscillations between +ve and -ve contrast can leave artefacts in the image
• CTF (contrast transfer function) describes how the system modulates the contrast of various spatial frequencies in the specimen
• CTF is mainly interested by lens abberation and defocus
• These can alter the phase and amplitude of the e- waves that interact with the specimen
• Need to correct images for CTF-induced distortions

20
Q

Phase plates as a way to generate contrast

A

Cause phase shift using a carbon disk in the column

21
Q

Describe the characteristics of a biological sample that would lead you to choose cryoEM as a structural technique

A

• CryoEM was traditionally used for big objects e.g. viruses and ribosomes
• Since DD detectors we can do smaller and smaller sizes
• Comfortably we can image >150kDa
• We can use a very small amount of material
• 2.4-4.5 microL material
• 0.01-0.11mg/ml conc
• X ray crys would need 10mg/ml
• Can visualise things in a membrane environment
• Can visualise in native environment

22
Q

Understand what limits resolution when imaging biological samples

A

• Heterogeneity in cryo EM is important
• We need to get pure samples for the image
• Usually prep by recombinantly expressing the protein
• Culture and lyse the cells
• Use affinity/size exclusion chromatography
• Assess purity in SDS-PAGE or activity assay
• Can negative stain (see later)
• Compositional heterogeneity is when distinct regions of the compound have different chemical compositions
• This can lead to variations in e- density which can lead to differences in image contrast
• Conformational heterogeneity – we want to limit number of different conformations of the compound to limit the signal to noise ratio
• Goal of sample prep to to balance preserving the native structure with limiting the signal: noise

23
Q

What is negative staining

A

• Protein is adsorbed to a carbon support
• Blot excess liquid and add a heavy metal stain to give amplitude contrast (difference in density)
• Heavy metals have high atomic number so scatter the e- more
• Use tungsten or uranium
• We aren’t imaging the object, we are imaging the stain surrounding the object
• Usually use a copper grid with wire mesh and a thin carbon film
• Initially very hydrophobic
• Apply a charge to the carbon film with a glow discharge machine
• Applies a -ve charge so sample with adsorb and liquid will distribute over the surface of the mesh
• Sample is loaded into the tip of a room temp holder
• Tip goes inside the column

24
Q

Advantages of negative staining

A

• Speed of screening – very quick image
• High contrast- because of high atomic numbers of heavy metals, doesn’t suffer from bad signal:noise
• Radiation hard- not sensitive to radiation as sample is dehydrated and gone so all that’s left is metal stain

25
Q

Disadvantages of negative staining

A

• Resolution limited – imaging a cast so limited by the grain size of the stain (20A)
• Protein distortion – can get distortions in how thick the material is

26
Q

Describe CryoEM as a sample preparation technique

A

• Sample transferred to metal mesh and excess material removed
• Sample forms a thin film across the holes in the mesh when its shot into ethane at -190 deg C
• Water vitrifies around the sample, which is then cooled by liquid nitrogen during the measurements in EM

27
Q

Types of ice

A

• Not all ice is the same
• Vitrous ice (good) is water that’s frozen in a glass-like state (transparent)
• Obtained by temp and speed at which you freeze the substance
• Crystallin ice = bad, shows up like leopard spots and scatters e-
• Can get ice contaminants in bad freezing conditions
• Can also get surface contaminants due to moisture when loading the sample after freezing it

28
Q

Issues with getting the particles into the ice

A

• Getting the particles into the ice is a major challenge
• Ideally want a random distribution of orientations
• Forces are created at the air/water interface as air is hydrophobic and water is hydrophilic
• This can change the orientations/distribution of objects
• Can introduce a support to adsorb the particles but the particles may aggregate/ have preferred orientations/ be denatured by the hydrophobic air
• Using a support helps control protein adsorbption
• Used to be a carbon film it carbon caused extra scattering so bad signal:noise
• Now use graphene support (single crystal of carbon lattice) which is transparent to the e- mean so wont scatter e-
• Changing the strength/time of the glow discharge machine controls the charge of the graphene so controls how much protein adsorbs and can control conc of protein on grid

29
Q

Understand how to reduce impact of radiation damage to the sample

A

• Inelastic scattering causes ionising radiation which destroys bonds in biological material
• Increasing exposure/ number of exposures increases the radiation damage so high resolution info is destroyed
• We do low dose imaging
• Low e- doses at low magnification to find the area we want to take pictures of
• Focus area is near where the info we want is
• Radiation is only happening at high magnification (over a small area) near, but not on, where we want the image
• We apply these parameters to our image
• Parameters include refocus and roundness of beam
• We don’t want too much refocus or we get artefacts
• Then we use this info when we shoot the beam at our sample
• Direct e- detectors detect subtle scattering so we can use low doses, also takes fast exposures
• Take many frames
• Earlier frames have less damage so can have more weighting when we align

30
Q

Describe how 2D EM images are related to the 3D sample observed

A

• 2D images are projections of a 3D object
• 2D image can be ambiguous so you need many orientations and projections to be collected
• Goal of single particle analysis is to collect lots of single particle images
• Need 10s to 100s of thousands to get high resolution 3D structure
• Individual particles are very noisy due to transmitted and in elastic e-
• To harvest high resolution info and enhance the signal:noise you average all the pictures
• You also need different orientations to get accurate 3D structure
• Using Fourier transforms is essential in computer image development
• It is a conventient mathematical representation of the image
• Can use it to do mathematical computations in Fourier space then use the inverse to go back to the image

31
Q

• Projection theorem

A

• 2D projection is a central slice through thr 3D Fourier transform of the object
• Each 2D Fourier transform represents a unique particle as they are all in random orientations
• We need to relate each orientation to each other to compile them
• Then you can inverse Ft to get the structure
• Relating the images to each other relies on knowing the orientation
• 3 parameters: initial position, translation, rotation
• If you misalign them you get an average which isnt a true representation of the sample
• Heterogeneity in the sample would give artefacts and composites in the image
• Noise in the image is lowered by averaging
• Averaging can also cause errors if you have different conformations of the protein
• Need to separate and classify

32
Q

Protocol for single particle analysis to solve a structure

A

• Protein purification – want homogeneous sample
• Negative stain
• Generate initial model
• Do cryoEM on pure protein sample
• Collect images on microscope
• Many frames on fast exposure to create a stack of images
• Need to align the images in the stack

33
Q

Motion correction

A

• Flash freezing puts strain on the ice
• Tension is released when the sample is irradiated so particles can rotate
• Need to correct for it or image will be blurry

34
Q

Defocus estimation and CTF correction

A

• Objective lens introduces artefacts which are taken into account in the CTF
• CTF is oscillations of positive and negative contrast
• We find focus then take the image under focus
• Need to know CTF of images and individual particles to correct so everything has +ve contrast
• If we didn’t correct we would have distortions
Defocusing - when the microscope is deliberately defocused, the objective lens is adjusted to intentionally place the image plane in front of or behind the specimen. This results in a blurred image

35
Q

Particle selection

A

• We use lots of different automated software to separate out noise and contaminants from actual particles
• You will always have some contamination you need to clean up

36
Q

Particle classification

A

• Happens in 2D
• Don’t need to know relative orientation yet
• Separate real particles from ice contaminants/broken/denatured particles

37
Q

Projection matching

A

• We use the initial model/ reference image
• Can rotate reference by a certain angular increment and can calculate a projection with a known rotation
• Then you find the best matching raw 2D image and can assign the angle

38
Q

Reconstruction of the image

A

• Compile the 2D images to make 3D image
• Iterative process to always improve the model that feeds into the next cycle

39
Q

Classification

A

• Refined model still could be a mixture of conformations
• Can classify different states in the computer using 3D classification methods from machine learning tools

40
Q

Map validation

A

• You will get a lot of different structures
• DD improved signal: noise
• Detergent micelles contribute a lot to noise
• CCD scintillator was a big source of noise
• Should see secondary structure in the map that matches the model
• The iterative refinement process can lead to model bias
• Aligning noise to the model to match what it was
• Can create a phantom shape