Data Refinement for Macromolecules

Open ccp4/ccp4i and create new project

• • Click "Change Project", then "Add/edit project"

  • name "exp_6568" and choose the user directory           **note: this is an example experiment number. You will use whatever number Crysalis Pro uses for your experiment

Scale intensities from data

• • Click menu in upper left corner, choose "Data Reduction and Analysis"

  • Click "Scale and Merge Intensities"

    • Job title: "scala"

    • Run "Ctruncate" to output Wilson plot and SFs after scaling; output single MTZ file

    • MTZ input "exp_6568.mtz" (from Crysalis Pro)

    • MTZ output: "exp_6568_scala1.mtz"

    • Under "Define Output Datasets", type Crystal "exp_6568" belonging to project "exp_6568" Dataset name "exp_6568"

    • Click "Run", then "Run Now"

  • Check quality of data by double-clicking on scala, then "Show Summary"

    • Multiplicity should be more than or equal to 3

    • R-merge should be less than 10%

    • Mean signal-to-noise ([I]/sd[I]) should be more than 10

Refine data

• • Click on menu in upper left corner, choose "Refinement"

  • Click "Run Refmac5"

    • Job title: "refmac-rigidbody"

    • Do "rigid body refinement" using "no prior phase info"

    • MTZ input: "exp_6568_scala1.mtz"

    • MTZ output: "exp_6568_refmac1.mtz"

    • PDB input: "1JJ0_apo.pdb"             **note: this is an example pdb file. 1JJ0 is lysozyme with sucrose bound in the active site, apo means without water**

    • PDB output: "1JJ0_apo_refmac1.pdb"

    • Under "Refinement Parameters", Set resolution range from minimum [blank] to 3.00" (so, just set to 3 angstroms)

      • Also, make sure "Exclude data with freeR label..." is unchecked. Otherwise, an error will occur.

    • Run Now

  • Double-click on job, show log graphs, look at R-factor analysis, stats vs. cycle

    • Want this to be under 50% at this point

  • Click "Run Refmac5"

    • Job title: "refmac-restrained"

    • Do "restrained refinement" using "no prior phase information"

    • MTZ input: "exp_6568_scala1.mtz"

    • MTZ output: "exp_6568_refmac2.mtz"

    • PDB input: "1JJ0_apo_refmac1.pdb"

    • PDB output: "1JJ0_apo_refmac2.pdb"

    • Set resolution range from minimum [blank] to 2.00"

    • Run Now

Written up by John Y. Lee, June 2014