Fan Lecture 5

Question 1

What is MIR?

Answer 1

Multiple Isomorphous Replacement - multiple heavy atom derivatives

Question 2

Describe the steps to do MIR.

Answer 2

1) Obtain a complete data set with the protein crystal
2) Soak the protein crystals with a reagent containing a heavy atom without perturbing the native structure of the protein.
3) locate the positions of the heavy atoms by difference Patterson map. When a heavy atom is added to the protein in a specific position, its atomic structure factor will change each structure factor of the protein by a small amount
4) Repeat step 2 and 3 to obtain a second derivative and that must bind to a different site in the protein.
5) Combine phases from both heavy atoms to provide initial estimates for sufficient phases to calculate an initial electron density map of the structure

Question 3

Why do you need multiple heavy metal derivatives?

Answer 3

1) Increased phase information, improving the accuracy of electron density maps
2) Cross-validation: increasing confidence in the resulting crystal structure
3) Helps resolve phase ambiguities and increases the likelihood of successful structure determination

Question 4

What is the requirements for MIR to work?

Answer 4

1) Isomorphous: no changes in protein structures and crystal packing
2) Measurable changes in diffraction intensities
3) Enough resolution (enough data to find out the heavy metal phases/positions)
4) At least 2 heavy metal derivatives with the metals bound at different sites

Question 5

What is molecular replacement?

Answer 5

Using a known structure with initial estimates of the phases for a new structure.

Question 6

Give an example when you may use MR to solve a new structure.

Answer 6

Examination of structural changes induced by binding an inhibitor or substrate analogue to a protein having a known structure if the ligand is soaked into an existing crystal

Question 7

What happens if the phasing model is isomorphous?

Answer 7

You can compute initial p(x, y, z) from the amplitudes from the new protein and the phases of the model IF the known protein crystallizes in the same space group and has the same orientation in the crystal

Question 8

What happens if the phasing model is isomorphous?

Answer 8

The structure of the phasing model must be superimposed on the structure of the unknown protein and phases must be calculated from the properly oriented model.

Question 9

What is Patterson function?

Question 10

Why does Patterson function have no phases?

Answer 10

It’s based solely on the magnitudes of electron density peaks, without regard to their relative positions in space.

Question 11

For a molecule with n atoms, how many peaks in its Patterson map?

Answer 11

Number of peaks = n(n-1)/2