1. Stony Brook Integrative Structural Biology Organization
Introduction to Molecular Replacement
*Adopted from Eleanor Dodson, MRC LMB
TBD
LSB 434

2. Molecular Replacement Background
Briefly talk about Patterson function, Rotation, Translation search
When can you use molecular replacement
Some advantages/disadvantages of molecular replacement
When do you know you have the right solution?

3. Patterson Function
Patterson function can be calculated from just the diffraction data (intensity measurements)
Patterson function is the convolution of the electron density on itself i.e. ρ times ρ*
The Patterson map can tell us where atoms are relative to one another

4. THE VECTOR MAP OF TWO ATOMS
What is the complete set of vectors between two atoms?
There are four vectors, two equal and opposite interatomic vectors and two self vectors.
The vector map has a large peak at the origin and two lower peaks on either side of it, separated from the origin by the distance between the two atoms.
Vector atom 1 to atom 2
Vector atom 1 to atom 2
Vector atom 1 to atom 1
Vector atom 2 to atom 2
Vector map

5. THE VECTOR MAP OF SOME ATOMS
We can generate a vector map of a molecule by putting each atom in succession at the origin
molecule
Patterson

6. ROTATION FUNCTION First, consider the model Patterson
We put the model in a large P1 box and calculate the Patterson from the structure factors of the model in the P1 box.
model in large P1 box
ROTATION?
“search” model
Patterson of model in large P1 box
Clusters separated by P1 cell dimensions

7. ROTATION FUNCTION
The Patterson of our unknown structure contains self-vectors and cross-vectors, but because the cell was large, the self-vectors and cross vectors are well separated from one another.
self vector
cross vector

8. ROTATION FUNCTION
Just as we generated the Patterson for our model in the first orientation, we can generate the Patterson for the model in any orientation in any sized box.
model in same large P1 box
in different orientation
Patterson of model in large P1 box
in different orientation

9. ROTATION FUNCTION
When the models are in different orientations the Pattersons will not match one another. = X

10. ROTATION FUNCTION
However, when the second model is in the same orientation parts of the Pattersons will match one another, and we can “solve” the rotation function for the model. =

11. Steps in Molecular Replacement
Template Selection
Rotation Search
Translation Search
Calculate Initial Phases

12. When can you use Molecular Replacement
There is a known structure of a related protein
>30% sequence identity

13. Advantages/Disadvantages of MR
Model Bias
Generates an initial model for refinement
Easier to obtain phases by MR than experimentally

14. Do I have the right solution?
Check the map (Cα backbone follows density, sidechain packing)
Correct number of molecules in ASU
Different programs have different scoring functions
R-factor, Correlation Coefficients

15. Molecular Replacement Tutorial
PDB: 3NDO
Deoxyribose phosphate aldolase from Mycobacterium smegmatis
227 residues (~23 kDa)
Very high resolution data (1.25A)
Diffraction images available at proteindiffraction.org

16. Steps for Molecular Replacement
Identify homologous structures
Prepare search model* (Chainsaw)
Run molecular replacement (Phaser or Molrep)
Do you have the correct solution?
Structure Refinement

20. ClustalX

23. CCP4- Chainsaw
Manually truncate 3ng3 to monomer

24. ExPASy ProtParam

26. Matthews Coefficient
Matthews determined the average solvent content of protein crystals (~42%), so we want contents of ASU to give us ~50% solvent

28. Look at solution

29. Refinement