Electron Microscopy

Question 1

Advantages of cryo-EM

Answer 1

1) native environment (X-ray crystallography disadvantage: does not reflect biochemically functional buffers)
2) Visualize relevant conformations (not constrained by crystal packing)
3) not much material (μl) – [ conc ] as low as tens of nM

Question 2

Electron microscope advantages

Answer 2

1) Ernst Ruska 1986
2) v. high energy as waves (wavelength smaller than interatomic distances)
3) no diffraction limit as w/ light m
4) e can be focused by magnetic lenses
5) Images more powerful than diffraction patterns (no need to solve phase problems)

Question 3

Electron microscope disadvantages

Answer 3

1) vacuum to avoid unwanted scattering of ē (all water sucked out of sample)
2) High energy ē cause ionizing radiation = destruction of bonds (destroys sample when imaging)
3) limit exposure of sample to electron beam to avoid damage (collect noisy image)
4) samples move when irradiated even though frozen in ice (get blurry picture)

Question 4

2017 Nobel Prize chemistry

Answer 4

1) Jack Dubouchet - Vitrification method
-place aq. solution on cryo-EM grid
-sample distributed across grid; excess liquid removed
-sample plunge frozen in liquid ethane cooled to liq. N temp (-196º); dissipates heat fast -> don’t end up with crystal + ice, instead -> vitrified ice to image molecules inside

2) Frank’s Image analysis to 3D structures
-Worked out how to use computational methods to align/classify different projections and use Fourier analysis to create 3D reconstruction of individual object

3) Henderson’s vision for high-resolution cryo-EM
-technology development underpinning direct electron detector (can detect at e level)l

Question 5

Resolution revolution

Answer 5

1) Objects smaller & more complex; can be solved to a higher resolution
2) tech innovations + software developments enable resolution of structures (molecular information, atomic resolution information, information that describes the chemistry of the interactions

Question 6

Relationship between wavelength and frequency

Answer 6

wavelength and frequency are reciprocal (F = 1/λ)

Question 7

Fourier transform

Answer 7

1) convenient mathematical relationship used in image processing to speed up calculations
2) representing wave in F not λ ie. in “reciprocal space”
3) calculates component sine waves of composite wave
4) inverse FT puts components back together to get original composite wave
5) shift up= sine wave w/infinite λ
-Sine wave with F = 0 is the DC component
-Has a given amplitude and it raises/lowers the level of all other waves
6) FT needed because e lenses introduce severe artefacts into images and need to be able to computationally correct these

Question 8

2D waves

Answer 8

1) Have 1 phase shift; 1 amplitude; and frequency/wavelength is a multidimensional vector
2) Images are very complex composite 2D waves
3) FT of 2D wave in a 2D plot where each pixel represents a specific discrete 2D vector
-Eeg. 2, 0 is the vector described; -2, 0 is mathematically equivalent (Friedel pair)
-0, 0 is the DC component; DC component has appreciable amplitude because it goes from 0 to 1 (entire wave has been elevated)
-Other waves are represented in FT but have A = 0

Question 9

Phases vs amplitude

Answer 9

1) Phase shift of each 2D sine wave makes up the image
2) The relative positioning of each one is crucial to accurately represent final image
3) Amplitude is less important
4) Problem in crystallography: lack the phases

Question 10

3D waves

Answer 10

-Can represent 3D density in terms of lots of 3D sine waves

Question 11

Light vs TEM microscope

Answer 11

1) Similarities: source, condenser lenses, type of image detection
2) Differences: air vs vacuum

Question 12

Rayleigh criteria

Answer 12

1) describes distance necessary to resolve 2 discrete spots
2) defines theoretical resolution limit of imaging technique
3) D = (0.61 x λ)/NA

Question 13

Why do we need electrons?

Answer 13

-Resolution depends on λ
-v. short ē λ = v. high resolution (~0.0015nm)

Question 14

What happens when e encounters atom

Answer 14

1) Most ē get transmitted straight through and don’t interact w/sample (~80%)
-Affects signal:noise ratio
2) ē can be backscattered; not recorded by detector
3) ē can be scattered elastically or inelastically
-Elastic: energy is conserved
-Inelastic: energy loss when ē encounters sample –in form of radiation damage (3x higher) & contributes to noise in image

Question 15

E microscope structure

Answer 15

1) Source
i) Crystal heated up under vacuum; emits ē
ii) thermal ionic emission sources = less coherent system (e.g LaB6 filament, tungsten filament least quality)
ii) field emission gun (new) = very coherent ē source =highest quality
2) Condenser lens
-i) Responsible for brightness and coherence of beam
ii) Strength of magnetic field focuses beam
3) Condenser aperture
i) Removes highly scat. ē allowing more coherent beam to pass through
ii) Removes spherical aberrations of lens
4) Sample in sample holder - Beam is scattered
5) Objective lens - focuses beam
6) Objective aperture
i) Increases image contrast
ii) Removes ē that contribute to noise/reduce contrast
7) Projector system -Amplification/ magnification of the image
8) Image detecting
i) Direct electron detectors( v. fast frame rates, instant feedback, high sensitivity)
ii) Skip e to light conversion by scintillator in CCD camera (loss of info; delocalisation of signal; decreased sensitivity of readout)

Question 16

Particles move in the ice

Answer 16

1) e beam releases some pressure when irradiating sample
2) speed + sensitivity of detectors can correct & align individual movie frames to enhance S:N = sharper image = correcting for beam induced movement

Question 17

Contrast

Answer 17

1) Measured by the difference in scat vs. unscat waves
2) 2 types: Amplitude and phase contrast (more important)
-Due to the wave particle duality of electron
3) other ways to generate contrast:
-Phase plates: thin metal films
-Benefit in cryo-e tomography

Question 18

Amplitude contrast

Answer 18

1) 3 criteria generate A contrast: diff in thickness, density, lattice pattern
-v. thin cryo-EM sample -> not much diff in thickness
-Similar atomic numbers -> not much diff in density (negative stain EM -> high Ar heavy metals)
2) Diffraction mode in cryo-EM= look at 2D crystals or tiny 3D crystals -> A contrast more important
3) X-rays: denser material scatters x-ray more strongly than softer tissue

Question 19

Phase contrast

Answer 19

1) due to wavelike properties of ē
2) Plane waves move down column of microscope, encounters sample, scattered by sample, complex wave phase shifted 90º behind unscattered wave (planar wave + spherical wave)
3) Each scat. sample interacts constructively/destructively w/other scat waves

Question 20

Detector

Answer 20

1) Detects intensity (I = A^2)
2) Sum of unscat + scat waves
i) Scattered wave undergoes additional phase shift bringing it back into focal plane for imaging
ii) Second phase shift = +90 = total 180º shift from incident beam
iii) Scat wave contributing maximally in opposite direction
iv) A less than unscat wave = negative contrast
3) Each different angle of scattered wave has different resolution ranges -> additional phase shifts
i) +90º = 270º -> changes unscat wave minimally (zero contrast)
ii) +90º = 360º ->contributing maximally in same direction as unscat wave (further into positive contrast)
4) No contrast for v. low scat angle; v. close to unscat beam; v. little phase shift (negligible); does not contribute to difference in A

Question 21

Lenses

Answer 21

1) diff strengths
2) Have aberrations (more apparent at higher scattering angles)
3) e bent more strongly at periphery

Question 22

Focusing

Answer 22

1) Applies additional phase shift = changes contrast
2) Changing focus on lens = changes oscillation between +ve and -ve contrast as a function of resolution
3) By changing focus:
i) silencing contributions to different waves at different spatial frequencies/resolution ranges
ii) Delocalisation of signal
iii) Distortions propagate in how you interpret images

Question 23

Sample requirements

Answer 23

1) Size
i) can see large macromolecular assemblies not accessible to other techniques
ii) ~150kDa = bigger is fine; smaller more challenging= less dense= scatters ē less (~60kDa for high resolution)
2) Quantity
-Sig less material than other techniques
3) Environment
i) look at protein in more native environment e.g. biologically relevant buffers, membrane proteins, etc
ii) cellular imaging
ii) complex imaging e.g. flexible dynamic assemblies, heterogenous assemblies, etc

Question 24

Heterogeneity in cryo-EM

Answer 24

1) Allows to solve structures that are more flexible in different conformations
2) Does not need absolute homogenous material packed in crystallographic arrays

Question 25

Goal of sample preparation

Answer 25

Balance between S:N and preserving native structure

Question 26

Negative stain

Answer 26

1) Apply sample which absorbs to C support
2) Add heavy metal stain
0Surrounds sample; image cast left behind from dehydration
3) Amplitude contrast is significant
4) EM grid has hydrophobic C; apply -ve charge
5) Drop method for negative staining
-Apply sample; wait for it to absorb; blot away; wash steps with metal stain
6) Hazardous process
- radioactivity associated
- dispose of stains appropriately
7) Advantages:
-V. fast screening sample (~2mins)
-High contrast; see small particles
-Radiation hard; cast not damaged by e beam
8) Disadvantages:
-Limited resolution; imaging cast not protein (limited by grain size of stain; typical ~20Å)
-Protein distortion if stain does not fully embed protein

Question 27

Vitrification method

Answer 27

1) Sample suspended in v. thin liquid film in random orientations  frozen
2) Not all ice is the same
i) Depends on temp and speed (where you are in phase diagram)
ii) Slow freeze = leopard print in background
iii) Ideal ice = glass-like to see through sample
3) Grid has square patch w/regular holes in C film
4) Charge distributes across C and helps aq. solution spread due to surface tension
5) Liquid goes into holes in all random orientations = thin meniscus
6) Reality: potential bulk particle behaviour at air-water interface
-Mismatch between hydrophobic air and hydrophilic water
7) Proteins will orient relative to air-water interface; sometimes they denature; can lead to aggregation
8) reduce impact by putting continuous support underneath holes to control protein adsorption e.g. thin layer of crystalline graphene
9) speed up vitrification process to minimise impact of air-water interface

Question 28

What to control when loading sample

Answer 28

Humidity and temperature (liquid N)

Question 29

Minimising radiation damage

Answer 29

1) se low-dose imaging
2) Focus on C support nearby
3) Take low M view of grid for good area then take subsequent higher M images for focus and exposure
4) K3 direct electron detector has helped minimise rad damage
-High sensitivity = can take images at lower exposure/dose rate
-Can achieve resolutions at sidechain level

Question 30

Factors influencing orientation in ice

Answer 30

1) Adsorption to grid support
2) Forces at air-water interface
3) Buffer they are in (e.g. detergents)

Question 31

2D images

Answer 31

1) Projections of a 3D object
2) Collect projections at lots of different orientations

Question 32

Goal of single particle analysis

Answer 32

1) Collect wide field of view on microscope then isolate individual particles by cropping out of image
2) S:N = v.poor
3) Goal to collect +1mil particles to get averages to enhance S:N

Question 33

Projection theorem

Answer 33

1) 2D projection is a central slice through the 3D FT of the object
2) Fill up box with all diff projections in diff orientations to give undistorted and complete view of 3D object in F space; then get original 3D object in real space

Question 34

Direct e detector

Answer 34

1) Collects 2D projections of object frozen in ice
2) Calculate 2D FT of 2D projection
3) Figure out orientation relative to all other 2D projections and all FT of 2D projections because each represent central slice in 3D FT of object
4) Want to sample all space to not end up w/distortions, missing views, etc

Question 35

Determining orientation paramters

Answer 35

1) Translations (x, y) and rotations (defined by Euler angle space)
-No z because it is 2D
2) If articles misaligned -> av. does not reflect real object -> need accurate orientation parameters

Question 36

Averaging over single particles

Answer 36

1) Isolate individual particles
2) av. over things not the same = mixture
-See partial density of things
3) Bayesian and machine learning algorithms separate out and classify noisy images not possible by biochem (isolate diff groups, conformations, states, etc)

Question 37

Overview of EM process

Answer 37

1) Protein purification
2) Negative stain to optimize purification
3) Initial model
-Get initial structure (low resolution)
-Rotate in computer
-Create catalogue of reference projections
4) Cryo-EM image
5) Isolate individual single particles (distinguish from contaminants)
6) Calculate and measure contrast transfer function (CTF)
7) Particle alignment and classification
-Classified based on orientation, composition, heterogeneity; av. to enhance S:N
8)Use projection theorem to create final structure
-Projection matching: compare known orientations from model to what’s been experimentally collected in the microscope
-calculate and align for the best match

Question 38

First step: motion correction

Answer 38

1) Collect movie frames that give fast frame readout
2) Align & correct for beam induced motion
3) Beam-induced motion = property of illumination for the electron beam (particles can move after being irradiated; need to capture in fast frames how they move in ice)
4) Conformational flexibility = proteins are dynamic and flexible; have arm that can reach out and grab object and imaging samples at all diff conformations of that biological process

Question 39

Defocus estimation and CTF correction

Answer 39

1) At different resolution ranges:
-Oscillating between +ve and -ve -> have to correct for
-Have zero info at spatial frequencies
2) Take images at diff defocus ranges:
-If only one, all images would be missing info at same spatial frequency/resolution -> distortions in object
-av. images to fill in gaps of missing info from CTF

Question 40

CTF

Answer 40

1) CTF envelops all distortions that come with focusing w/objective lens
-Spherical aberrations
-Distortions from +ve and -ve contrast
2) Must correct distortions before interpreting 3D structure
3) CTF is changing as a function of resolution

Question 41

Challenges

Answer 41

1) Noise being confused for signal, so noise builds up
-Expect to see real internal and 2º features of protein (e.g. helices, strand separation, etc); if not, averaging junk and should get rid of
2) Map validation: who has the right model?

Question 42

Model bias

Answer 42

1) take noise; align to reference; start to see build-up of noise to reinforce image that was in reference
2) helps recognise what has gone wrong in averages