Hi all, I'm trying to run InterfaceAnalyzer in the "classical" and xml script modes. In the "classical" mode I ran: > InterfaceAnalyzer.linuxgccrelease @flags_IL2_Q74P_2 ... with this flags: -in:file:l ../IL2_IL2Ra_new_0001.pdb -in:file:fullatom -compute_packstat true -tracer_data_print true -out:file:score_only score.sc -use_resfile yes -resfile IL2_Q74P.resfile -fixedchains A B -pack_input false -pack_separated true -add_regular_scores_to_scorefile true -use_jobname true -database /home/almeida/Softwares/Rosetta/rosetta_3.4/rosetta_database The run crash with this error: core.chemical.ResidueTypeSet: Finished initializing fa_standard residue type set. Created 6225 residue types core.import_pose.import_pose: PDB File:ATOM not found! ERROR: Cannot open PDB file "ATOM" ERROR:: Exit from: src/core/import_pose/import_pose.cc line: 184 On the other hand, I used rosetta_scripts application, running: > rosetta_scripts.default.linuxgccrelease @flags_IL2_Q74P_1 ... with the following flags: -parser:protocol mutation_script_IL2_Q74P.xml -s ../IL2_IL2Ra_new_0001.pdb -ignore_unrecognized_res -database /home/almeida/Softwares/Rosetta/rosetta_3.4/rosetta_database -out:path:pdb ./ -out:path:score ./ -nstruct 1 ... and a segmentation fault arise: core.pack.task: Packer task: initialize from command line() ./run_Interface_Analyzer_Q74P_1.sh: line 6: 23713 Segmentation fault (core dumped) rosetta_scripts.default.linuxgccrelease @flags_IL2_Q74P_1 The .xml script (mutation_script_IL2_Q74P.xml.txt) and the resfile (IL2_Q74P.resfile.txt) are attached. Thanks in advance for the help!! Regards
First, your top and bottom questions relate to totally different protocols for different purposes. The top is InterfaceAnalyzer, which will give you some information about the quality of an existing interface. The bottom is a forced-mutation protocol to make a single Q74P mutation.
For the top error, you need to use -in:file:s for a single PDB, not -in:file:l. The error is because it is trying to interpret the PDB as a list of files, thus the file "ATOM" (the first thing in your PDB) is not found. Try that first.
For the bottom error, I suspect the error is in your DDG filters. You have these notes in the XML file:
" Here we add two delta G filters to calculate the delta G of binding before and after mutations are made.
We specify a jump number of 3 because want to calculate the delta G of binding the ligand, which is chain D
in the given PDB file."
Does that hold true for your intended purpose? If not, what are you trying to do here?
For the first error I used -in:file:s and the protocol write the score file for the native protein without any mutation. I realized that in the online manual of Rosetta 3.4, for InterfaceAnalyzer say that:
"-use_resfile (bool) - warns the protocol to watch for the existence of a resfile if it is supposed to do any packing steps. (This is normally signealed by the existance of the -resfile flag, but here the underlying InterfaceAnalyzerMover is not intended to use -resfile under normal circumstances, so a separate flag is needed. You can still pass the resfile with -resfile.) Note that resfile commands indicating design are ignored; InterfaceAnalzyer does repacking ONLY."
This means that the mutation flag is ignored?
For the second error, the objetive is to compute the DDG for native and mutant interfaces, using the XML script. I used this XML file as template for my problem. Originally the file set that:
"We specify a jump number of 3 because want to calculate the delta G of binding the ligand, which is chain D in the given PDB file."
I want to calculate the delta G of binding before and after Q74P mutation. My complex is formed by two chains, 1 and 2, where the chain 2 is mutated. The template use a jump number equal to 3, corresponding to chain D which is mutated. In my case I don't know what value to use.
Yes, any mutations indicated or allowed by your resfile will not actually occur. InterfaceAnalyzer reports only on the quality of the interface it is given. The resfile override is present to allow you to define for yourself how to do the packing in the bound and unbound states - perhaps you have some site you do not want repacked, etc.
You have two chains, so you have one jump between them. Use jump number 1.