Stony Brook Integrative Structural Biology Organization Introduction to
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. 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 P 1 box and calculate the Patterson from the structure factors of the model in the P 1 box. model in large P 1 box ROTATION? “search” model Patterson of model in large P 1 box Clusters separated by P 1 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 P 1 box in different orientation Patterson of model in large P 1 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 (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: 3 NDO • Deoxyribose phosphate aldolase from Mycobacterium smegmatis • 227 residues (~23 k. Da) • Very high resolution data (1. 25 A) • 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
Clustal. X
CCP 4 - Chainsaw • Manually truncate 3 ng 3 to monomer
Ex. PASy Prot. Param
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
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