Be careful here as anisotropic temperature refinement can make your obs:params ratio rather low, and hence you only want to do this if you have a lot of data.įinally in this section you can pick your Rfree set. For data down to about 2A isotropic temperature factors is normally a good idea, and if you have even higher resolution data you can change this to anisotropic temperature factors. The next line allows you to select B factor refinement which I would suggest NOT doing initially or if you have data worse than 2.6A resolution. If you have good high resolution data 0.3 is reasonable, however if you are having problems with an overly high Rfree or poor geometry, I would recommend values as low as 0.1. Thus I always deselect this box, and then try to type in a sensible value next to the word "Use weighting term". I have found that this generally puts too heavy a weight on the X-ray terms and thus gives rather high Rfree values at the end of a refinement. IMPORTANT - as a default the program next selects "Use experimental sigmas to weight X-ray terms". If you want to alter the resolution of the data that is refined you can do this by clicking the box next to "Resolution range from.". Initially 10 rounds of refinement should be OK, however sometimes you might need to increase this if your refinement is not converging (see below). The next section prompts you to give a "dataset name" which again is arbitrary, however after this you get to the most important box titled "Refinement parameters": Ignore the library line if you have yet to build a ligand into your model (see below if you have built a ligand already). Also worth watching is the tabs saying "FP" and "sigma" which should automatically pick up you structure factor amplitude and error columns from your mtz file once you have told the program which mtz file to use. The program should then automatically generate names for the new files it will create once the it has run the refinement job (MTZ out and pdb out), however you should check the paths to make sure the files are going to be saved in the correct place. TURBO you need to select the "Generate weighted difference map files in CCP4 format" option.Īs with other ccp4i programs you are next prompted to give the program your input files which in this case will be your experimental data as an MTZ file (MTZ in) and your molecular replacement solution as a pdb file (PDB in). However if you need to create map files for other graphics programs (e.g. If you are going to be using Coot for your model building I would ignore these. Next the program gives you some check boxes titled "Input fixed TLS parameters". You can also select TLS (translation, libration and screw-rotation) with restrained refinement, however I would suggest running a couple rounds of normal restrained refinement before starting to play with TLS. If you are just beginning a refinement round you want to select "rigid body refinement" for your first refinement round, however after this you will probably need to be running restrained refinement. This can be selected by clicking on the "Do restrained refinement" tab which will give you the following drop-down menu: Starting at the top of the menu you need to give your job a title (something you will be able to remember), and then select the type of refinement you would like to perform. To open the program click on the yellow tab in the top left corner of the ccp4i GUI, select "Refinement" and then "Run Refmac5". It is written by Garib Murshudov at the University of York, and its web page can be found here. Refmac is a refinement program that comes bundled with ccp4i and is probably the easiest to use if you have just scaled and phased your data using ccp4.
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