Lecture 18: Electron Diffraction

Question 1

What is the process of micro crystal electron diffraction

Answer 1

Obtain crystals

Collect data

Obtain phases:
- search for the orientation and position of a similar molecule in the crystal

Calculate density from data and phases

Fit the model to the electron density

Refi e the model using the data

Do last three steps again and again

Question 2

What are the crystals for microED

Answer 2

crystals have to be very thin (160 angstroms in all directions)

Need 3D crystal but can do with 2D (just a single layer crystal, no translational symmetry)

Question 3

What are the wavelength ranges for X ray diffraction and microED

Answer 3

1 A for X ray crystallography to make images of atoms

Adding in electron beam (0.0251 angstroms) can collect data to extremely high res because smaller wavelength but things prevent that from happening

Question 4

Compare light and transmission electron microscopes

Answer 4

inverted microscope (source at the top in both cases)

Rays go throigh the object and to the detector

Source of electrons at the top and Beam goes down and through the sample

What matches is that there are lenses in light scope and in TEM :
- in X ray there are no lenses so you can’t focus beam like you can in TEM and light

Question 5

Explain the TEM

Answer 5

The electron source:
- is a field emmision gun (FEG) or thermionic emmision
- Electron source give the diff energy spread/ bandpass where FEG has tighter bandpass

• when electron beam hits the sample, path of electron beam changes by 2theta due to diffraction
• the electrons pass through the sample and lenses

Lenses between beam and detector :
- you can switch beteeen Collecting images and collecting diffraction pattern
- Purple dots are diffraction spots, Green lines are diffraction rays imaged by detector
- then you can record diffraction as a video on the detector

Question 6

What is the characteristics of samples in TEM

Answer 6

The electrons behave as waves :
- scattered in all directions and interfere constructively and destructively
- but we want only one scattering event in the sample

So Need thin sample:
- dont want many scattering events, don’t want one ray to interact with multiple atoms which happens if you have a thick sample

The diffraction pattern is related to atomic structure

Crystalline material gives discrete spots in diffraction pattern

Question 7

Explai nthe 2D crystals in micro ED

Answer 7

in 2D crystals there is no stacking in the z direction, so in diffraction pattern you have discrete pattern along x and y but in z there are just lines

Can tilt the sample to get diffraction across the lines and get diff diffraction patterns

The diffraction pattern gives the Average of the signal from all of atoms in the path of the beam

Question 8

Explain how samples are prepared for microED
Issues with liquid Ethan and nitrogen

Answer 8

Same as for cryo em:

Small volume of sample added to the grids

Blot off excess

Plunge grid into liquid ethane

X ray crystal we use cryoprotectant and put into cold nitrogen but For this we need liquid ethane and liquid nitrogen keeping the ethane cold

Problems:
- liquid nitrogen can go into the air and cause asphyxiation
- liquid Ethane is more flammable and volatile
- both are very cold

After vitrified, store the grid at temp of liquid nitrogen (77K)

Don’t get fast enough heat transfer away from the grid with liquid nitrogen on its own that why liquid ethane is used.

Question 9

Contrast braggs law in X ray crystallography with microED

Answer 9

The wavelength is shorter, and because of this there is barely any change in the angle of diffraction.

Incident angle is short so 2theta is still short

Question 10

Explain the ewalds sphere for microED

Answer 10

Becuase wavlngth is 0.021 and the radius of the sphere is 1/wavelength

1/0.0251 is a very big ewalds sphere, almost flat

so lots of spots in the diffraction pattern since sphere is huge and can collect way more points

Question 11

Do you also rotate the crystal in TEM

Answer 11

Yes, have to rotate the crystal to get unrecorded reflections into diffraction conditions for x ray cryst

Have to do the same and rotate the microED crystal in the TEM scope, get way more reflections since big ewalds sphere

Question 12

How are phase found in TEM

Answer 12

Molecular replacement , same as in x ray crystallography trial and error

Question 13

How is density found in mocroED using TEM and what are the maps

Answer 13

The electrons are negatively charged and scattered via coloumbs forces:
- they interact with both the postive atomic core and negative electrons around the core

Maps are not electron density but maps of coloumb potential

Then a contour map is calculated to make the coloumb potential interpretable

Question 14

How do the refinement diff in microED

Answer 14

each atoms has three things:
- atomic position
- b factor
- occupancy

The scattering from the both core and the electrons now changes things:
- Difference is in fj, electron scattering factors (not atomic scattering factors anymore)

Question 15

How is the scattering factor in microED diff than x ray crystallography

Answer 15

In X ray:
- all you need is resolution from sintheta/lambda to figure out the scatteing of the atoms
- all atoms start at lower resolution with high scattering, fall off as you go to higher resolution

Electron scattering factors
- now Need to know the charged state of the atoms in mocroED
- Oxygen in diff state in TEM has diff resolution (o vs o-)

Question 16

Explain the protien in example 1 of microED

Answer 16

Structure of the toxic core of alpha synuclein from invisible crystals (crystal of dimensions smaller than wavelength of visible light)

This protien is the main component in Lewy bodies which are aggregates seen in Parkinson’s disease

Protien:
- has amphipathic N term and acidic c term
- Core NAC has 11 residues which is the key part in forming Lewy bodies
- Propensity to form beta strands

Question 17

Explain the crystals they got in example 1 of microED

Answer 17

visible crystals:
- had crystallized the diff subunits of the protien
- the subNAC core gave larger crystals whos structure could be solved by x ray crystallography
- the preNAC and NAC core structure were found by microED (invisible crystals)

Models were use to solve the phases via MR “invisible crystals” (MR)

Question 18

Explain the diffraction they got in example 1 of microED

Completeness in microED compared to XRD

Answer 18

Since it’s a small molecules and not a protien you see Less spot in the diffraction image :
- smaller unit cell in real space, larger unit cell in reciprocal space , not many spots

They Rotated the crystal by tilting

Can only go a certain distance (40-80 degreees), so you get less data in microED than x ray crystalography so less completeness than in x ray diffraction

Question 19

Explain the model they got in example 1 of microED using the difference map

Answer 19

Did a difference map of the NACcore and subNACCore search model

at the ends they have electron density with nothing in it:
- had to fill this in with residues

Also extra density throughout:
- is the other beta strand in the crystal because this core forms beta strands

Question 20

Explain the protien in example two of microED and how they did this

Answer 20

Combine mass spec and microED to discover enzyme-small mocleule complex:
- ACT bound to lysozyme

soak tiny crystal in either the pure ACT or inhibitor cocktail with ACT:
- do microED and see density for a ligand bound to lysozyme

Native mass spec identifies which of the compunds they could actually model into the electron density with the protien:
- take the soaked crystals and get mass estimates via mass spec
- Find the diff between the two masses and that diff matches the mass of ACT
- That’s how they know to fit ACT Into the density
- A way to screen a lot of compounds for their binding to their protien