Stony Brook Integrative Structural Biology Organization Introduction to Molecular Replacement *Adopted from Eleanor Dodson, MRC LMB TBD LSB 434
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? 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. times * The Patterson map can tell us where atoms are relative to one another
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. Vector atom 1 to atom 2 Vector atom 1 to atom 2 Vector atom 1 to atom 1 Vector atom 2 to atom 2
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 map 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
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
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 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
ROTATION FUNCTION When the models are in different orientations the Pattersons will not match one another. = X
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. =
Steps in Molecular Replacement 1. 2. 3. 4.
Template Selection Rotation Search Translation Search Calculate Initial Phases
When can you use Molecular Replacement There is a known structure of a related protein >30% sequence identity Advantages/Disadvantages of MR Model Bias
Generates an initial model for refinement Easier to obtain phases by MR than experimentally Do I have the right solution? Check the map (C backbone follows density,
sidechain packing) Correct number of molecules in ASU Different programs have different scoring functions R-factor, Correlation Coefficients
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 Steps for Molecular Replacement 1. Identify homologous structures 2. Prepare search model* (Chainsaw) 3. Run molecular replacement (Phaser or
Molrep) 4. Do you have the correct solution? 5. Structure Refinement ClustalX
CCP4- Chainsaw Manually truncate 3ng3 to monomer ExPASy ProtParam Matthews Coefficient
Matthews determined the average solvent content of protein crystals (~42%), so we want contents of ASU to give us ~50% solvent Look at solution
Refinement