Running ARP/wARP for Auto-Building (#4)

THIS IS AN $\alpha$ VERSION!
Well, just run warpNtrace.sh and see what happens.

This script uses the parameters from warp.par to perform cycles of iterative auto-tracing, model building and refinement. You probably may have a cup of coffee or a glass of beer while your protein is being auto-built. Do not get disappointed if the protein does not get traced completely ( AFTER ALL THIS IS AN $\alpha$ VERSION).

Look at the connectivity index which is output after every building cycle. If it is higher than 0.4 you have a good chance of improving it to more than 0.9, i.e. tracing almost the whole thing. If it starts much lower, and no usable chains are reported even after the first building cycle, you can start on the O manual ...

Here is a typical example of how the model for a 460 residues protein gets better and better, with a good (i.e. properly measured low resolution) native dataset (kindly provided by Peter Metcalf) extending just to 2.0 Å resolution:

Molecule 1:	20	chains,	247 residues, 1355 atoms.
Molecule 1:	20	chains,	319 residues, 1819 atoms.
Molecule 1:	17	chains,	395 residues, 2272 atoms.
Molecule 1:	16	chains,	390 residues, 2248 atoms.
Molecule 1:	14	chains,	405 residues, 2329 atoms.
Molecule 1:	10	chains,	416 residues, 2411 atoms.
Molecule 1:	11	chains,	418 residues, 2424 atoms.
Molecule 1:	14	chains,	428 residues, 2480 atoms.
Molecule 1:	10	chains,	435 residues, 2533 atoms.
Molecule 1:	12	chains,	434 residues, 2533 atoms.
Molecule 1:	9	chains,	435 residues, 2535 atoms.
Molecule 1:	10	chains,	442 residues, 2578 atoms.
Molecule 1:	9	chains,	444 residues, 2580 atoms.
Molecule 1:	11	chains,	443 residues, 2583 atoms.
Molecule 1:	11	chains,	441 residues, 2577 atoms.
Molecule 1:	11	chains,	442 residues, 2583 atoms.
Molecule 1:	11	chains,	438 residues, 2565 atoms.
Molecule 1:	8	chains,	448 residues, 2617 atoms.

Quite impressive, isn't it?

Tricks and Tips

• This script will first look for a file called warp_free.brk which it will use as a starting model. If this file doesn't exist it will try to build it's own initial model from the FoFOM.ext map (that is created after warp averaging).

• If you have a free atoms model from warp refinement without averaging (i.e. model_1_rc3.brk), copy it to files/warp_free.brk of your working directory.

• If you used warp averaging the script will look for the FoFOM.ext map and go on from there.
• If you have already good phases, you just could try to run warp_build.sh, which will create a free atoms model using that map. The output will be placed in the files/ directory as mir_build_1.brk. Copy this file to files/warp_free.brk and start from that one.

• If you start from a molecular replacement model, just copy it to a file called files/warp_free.brk.

• If you are not satisfied with the number of cycles you did, and you think there is additional room for improvement, just copy /autobuild_main.brk to /warp_free.brk (both in the files/ directory) and restart.

• A very important issue is what you do if you have known heavy atoms in the structure. Usually you know where the heavy atom is, and you want to include it in the refinement. You will have to manually insert the line relevant to that atom in the warp_free.brk file. Then the script will take care not to delete it during auto-building, etc. The line should look like this:
  

  ATOM   5578 FE   IUM Y   1       2.322  -0.095   9.294  1.00  5.14  26
  

  Take care that the IUM notation is used, a valid solvent chain is used (X,Y,Z or W) and the correct atomic number is given in the last column, else the atom will be ignored.

  For multiple atoms, simply edit multiple lines.

• The script arp_warp_setup.sh automatically sets the number of internal REFMAC cycles depending on which protocol you chose and on the resolution in the MTZ file. For the Heavy protocol, i.e. this application, the number of cycles are 1 for d less than 1.1 Å , 3 for 1.1 < d < 1.3 and 5 if the resolution d is between 1.3 and 1.5 Å . These values may well be slightly rubredoxin specific...If you feel your case might require different numbers, simply edit the warp.par file and change the rrcyc value. Oh yes, and make sure you know what you are doing.

• There are three possible command line options for warpNtrace.sh that can be used in any combination and order. The option final makes sure that main chain atoms are not chucked out. Giving a number on the command line will reset the sigma level for finding atoms. A third option is keepgeom which will prevent all the atoms from being set to waters before the first building cycle (default is opposite).

• Even if you have a descent starting map and good data (above 2.0 A) it can happen that after getting around 80% of the main chain in a few fragments, (connectivity index 0.8-0.9) you can never reach the so much desired 0.99 and a full trace (this is mainly in folds with long floppy disordered loops).

  The easiest thing to do is to run warpNtrace.sh final, which will disallow wARP to remove main chain atoms which might be in not so good density and might increase convergence. If this fails, we recommend to run the script as warpNtrace.sh final 3 which will reduce the sigma level for finding atoms to 3 (default is 3.5) which might help to find some more atoms in disordered regions.