March 26

Question 1

What is the flowchard for macromoleuclar crystallography?

Answer 1

1. Obtain crystals
2. Collect data
3. Obtain phases by molecular replacement ( search for orientation and position of similar molecule in crystal), isomorphous replacement (heavy atom soaked into crystal), or anomalous diffraction by incorporate heavy atom

For isomorphous and anomalous, need to locate heavy atoms. Then for all pathways calc the electron density from data and phases
Then cycle of fitting model to electron density, then refinement

Question 2

What is microcrystal electron diffraction (microED)?

Answer 2

It is when we use really small crystals, they may be 2D (so not 3D, ex: only have 1 cystal unit in Z, but it is flat and has multiple unit cells in x and y directions.

Use small wavelength beam of electrons that interact iwth both electrons and nucleus of atoms

Question 3

For electron source for microED, which one makes electrons with a greater energy spread (variation)?

Answer 3

Have thermionic emission, has greater energy spread than field emission gun.

Question 4

How is Ewald’s sphere different for microED?

Answer 4

As we use very small wavelength (0.025 A), we have sphere with 1/wavelength for radius, so LARGE radius. This causes the sphere to seem flat. Still rotate crystal to get lattice points on sphere to get recorded diffraction

Question 5

What do we use to solve phase for MicroED?

Answer 5

Use molecular replacement. Trial and error. First find orientation of molcule, then search for postion.

Question 6

What forces scatter electrons for MicroED? What density do we get as result/

Answer 6

Coulomb forces. Maps are therefore coulomb potential not electron density

Question 7

How are scattering factors diff for MicroED to XRD?

Answer 7

XRD we have at resolution of basically zero, we have the aplitude of scattering at the atomic number of the atom. Not for MicroED, we have negatively charged atoms at differnt points than other atoms, not based on atomic number

Question 8

Why might you see fewer diffraction points for microED depending on the sample?

Answer 8

If have peptide instead of protein, then you have smaller unit cells, less in these unit cells, have weaker diffraction

Question 9

If have 2 crystals of same space group and angles, how could you tell if they are isomorphous?

Answer 9

If they have same unit cell lengths

Question 10

Is Rmerge, Rwork, Rfree higher or lower for electron beam?

Answer 10

All are generally higher than XRD

Question 11

What is rule of thumb for Rwork in XRD, but not in MicroED?

Answer 11

It is that the resolution should be 10 x higher than the value of R work