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. ρ times ρ* 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. 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

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 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 (Cα 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 Identify homologous structures Prepare search model* (Chainsaw) Run molecular replacement (Phaser or Molrep) Do you have the correct solution? 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