PROSS程序设计

需要提前准备resfile和pssm文件

首先生成.cst文件: make_csts.sh <input_pdb> >output_filename

make_csts.sh input_example.pdb >example.cst
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第二步，生成约束文件

准备文件：refine.flags, refine.xml, example.cst(这个是上一步生成的)
refine.flags：

-use_input_sc
-extrachi_cutoff 5
-in:ignore_unrecognized_res
-chemical:exclude_patches LowerDNA UpperDNA Cterm_amidation SpecialRotamer VirtualBB ShoveBB VirtualDNAPhosphate VirtualNTerm CTermConnect sc_orbitals pro_hydroxylated_case1 pro_hydroxylated_case2 ser_phosphorylated thr_phosphorylated tyr_phosphorylated tyr_sulfated lys_dimethylated lys_monomethylated lys_trimethylated lys_acetylated glu_carboxylated cys_acetylated tyr_diiodinated N_acetylated C_methylamidated MethylatedProteinCterm
-linmem_ig 10 
-ignore_zero_occupancy false 
-s input_example.pdb # path to structure file 
-out:path:pdb pdbs/ 
-out:path:score scores/ 
-parser:protocol refine.xml 
-parser:script_vars res_to_fix=1A,2A,3A,4A # comma separated list of positions 
-parser:script_vars cst_full_path=example.cst # path to Rosetta CST file of CA atoms
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注意：（1）需要手动在此目录下创建两个文件夹(pdbs,scores)
           （2）修改对应的xml，cst以及pdb名称。
refine.xml

<ROSETTASCRIPTS>
	<SCOREFXNS>
		<ScoreFunction name="ref_full" weights="ref2015" >
            <Reweight scoretype="coordinate_constraint" weight="0.1" />
            <Reweight scoretype="res_type_constraint" weight="0.1" />
        </ScoreFunction>
        <ScoreFunction name="soft_rep_full" weights="soft_rep" >
            <Reweight scoretype="coordinate_constraint" weight="0.1" />
            <Reweight scoretype="res_type_constraint" weight="0.1" />
        </ScoreFunction>
        <ScoreFunction name="ref_no_pssm" weights="ref2015" >
            <Reweight scoretype="coordinate_constraint" weight="0.1" />
        </ScoreFunction>
        <ScoreFunction name="ref_pure" weights="ref2015"/>
	</SCOREFXNS>
    <RESIDUE_SELECTORS>
        <Index name="ress_fix" resnums="%%res_to_fix%%"/>
    </RESIDUE_SELECTORS>
	<TASKOPERATIONS>
        <InitializeFromCommandline name="init"/>
        <RestrictToRepacking name="rtr"/>
        <OperateOnResidueSubset name="fix_res" selector="ress_fix">
            <PreventRepackingRLT/>
        </OperateOnResidueSubset>
        <OperateOnResidueSubset name="not_to_cst_sc">
            <Not selector="ress_fix"/>
            <PreventRepackingRLT/>
        </OperateOnResidueSubset>
    </TASKOPERATIONS>

	<MOVERS>
        <AtomCoordinateCstMover name="fix_res_sc_cst" coord_dev="0.5" bounded="false" sidechain="true" task_operations="not_to_cst_sc"/>
        <PackRotamersMover name="soft_repack" scorefxn="soft_rep_full" task_operations="init,rtr,fix_res"/>
        <PackRotamersMover name="hard_repack" scorefxn="ref_full" task_operations="init,rtr,fix_res"/>
        <RotamerTrialsMinMover name="RTmin" scorefxn="ref_full" task_operations="init,rtr,fix_res"/>
        <TaskAwareMinMover name="soft_min" scorefxn="soft_rep_full" chi="1" bb="1" jump="0" task_operations="init,fix_res"/>
        <TaskAwareMinMover name="hard_min" scorefxn="ref_full" chi="1" bb="1" jump="0" task_operations="init,fix_res"/>
        <ConstraintSetMover name="add_CA_cst" cst_file="%%cst_full_path%%"/>
        
        

        <ParsedProtocol name="refinement_block">
            <Add mover_name="soft_repack"/>
            <Add mover_name="soft_min"/>
            <Add mover_name="soft_repack"/>
            <Add mover_name="hard_min"/>
            <Add mover_name="hard_repack"/>
            <Add mover_name="hard_min"/>
            <Add mover_name="hard_repack"/>
            <Add mover_name="RTmin"/>
            <Add mover_name="RTmin"/>
            <Add mover_name="hard_min"/>
          
        </ParsedProtocol>
        <LoopOver name="iter4" mover_name="refinement_block" iterations="4"/>
	</MOVERS>
    <FILTERS>
        <ScoreType name="stability_score_full" scorefxn="ref_full" score_type="total_score" confidence="0" threshold="0"/>
        <ScoreType name="stability_without_pssm" scorefxn="ref_no_pssm" score_type="total_score" confidence="0" threshold="0"/>
        <ScoreType name="stability_pure" scorefxn="ref_pure" score_type="total_score" confidence="0" threshold="0"/>
        <Rmsd name="rmsd" confidence="0"/>
        <Time name="timer"/>
    </FILTERS>
    
	<PROTOCOLS>
        <Add filter_name="timer"/>
		<Add mover_name="add_CA_cst"/>
        <Add mover_name="fix_res_sc_cst"/>
        <Add mover_name="iter4"/>
        <Add filter_name="stability_score_full"/>
        <Add filter_name="stability_without_pssm"/>
        <Add filter_name="stability_pure"/>
        <Add filter_name="rmsd"/>
        <Add filter_name="timer"/>
	</PROTOCOLS>
    
    <OUTPUT scorefxn="ref_full"/>
</ROSETTASCRIPTS>

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接下来输入命令

/home/unbuntu/rosetta2019/main/source/bin/rosetta_scripts.default.linuxgccrelease -database /home/unbuntu/rosetta2019/main/database/ -in:file:s input_example.pdb -parser:protocol refine.xml -parser:script_vars res_to_fix=1A,2A,3A,4A -parser:script_vars pdb_reference=input_example.pdb -parser:script_vars cst_full_path=example.cst -parser:script_vars cst_value=0.4 @refine.flags -overwrite
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pdbs文件夹里会生成一个input_example_0001.pdb文件，scores生成score.sc文件

Command line variables

1. path_to_db: path to rosetta database
2. input_pdb: the input structure/model in pdb formant only
3. fix_list: a list (number and chain) of all residues to restrict to minimization separated by commas. Hypothetical Example: 1A,2A,3A,4A
4. cst_file: a file with the input coordinates of all CA atoms in the input_pdb. see example.cst and use make_csts.sh to make one.
5. cst_val: the coordinate constraint weight in the score function. PROSS default is 0.4

运行design

准备design.xml文件

<ROSETTASCRIPTS>

    <SCOREFXNS>
		<ScoreFunction name="talaris_full" weights="talaris2014">
			<Reweight scoretype="coordinate_constraint" weight="%%cst_value%%"/>
            <Reweight scoretype="res_type_constraint" weight="0.4"/>
		</ScoreFunction>

		<ScoreFunction name="soft_rep_full" weights="soft_rep">
			<Reweight scoretype="coordinate_constraint" weight="%%cst_value%%"/>
			<Reweight scoretype="res_type_constraint" weight="0.4"/>
		</ScoreFunction>

		<ScoreFunction name="tal_no_pssm" weights="talaris2014">
			<Reweight scoretype="coordinate_constraint" weight="%%cst_value%%"/>
		</ScoreFunction>

		<ScoreFunction name="talaris_pure" weights="talaris2014"/>
	</SCOREFXNS>

	<TASKOPERATIONS>
		<InitializeFromCommandline name="init"/>
		<ReadResfile name="read_resfile" filename="%%in_resfile%%"/>
		<RestrictToRepacking name="rtr"/>
		<PreventResiduesFromRepacking name="fix_res" reference_pdb_id="%%pdb_reference%%" residues="%%res_to_fix%%"/>
		<SeqprofConsensus name="pssm_cutoff" filename="%%pssm_full_path%%" min_aa_probability="0"  convert_scores_to_probabilities="0" probability_larger_than_current="0" keep_native="1"/>
	</TASKOPERATIONS>

	<MOVERS>
        <PackRotamersMover name="soft_design" scorefxn="soft_rep_full" task_operations="init,read_resfile,pssm_cutoff,fix_res"/>
		<PackRotamersMover name="hard_design" scorefxn="talaris_full" task_operations="init,read_resfile,pssm_cutoff,fix_res"/>
		<RotamerTrialsMinMover name="RTmin" scorefxn="talaris_full" task_operations="init,rtr,fix_res"/>
        <MinMover name="soft_min" scorefxn="soft_rep_full" chi="1" bb="1" jump="0"/>
		<MinMover name="hard_min" scorefxn="talaris_full" chi="1" bb="1" jump="0"/>
        <ConstraintSetMover name="add_CA_cst" cst_file="%%cst_full_path%%"/>
		<FavorSequenceProfile name="FSP" pssm="%%pssm_full_path%%" scaling="none" scorefxns="talaris_full" weight="1"/>

 		<ParsedProtocol name="design_block">
			<Add mover_name="soft_design"/>
			<Add mover_name="soft_min"/>
			<Add mover_name="soft_design"/>
			<Add mover_name="hard_min"/>
			<Add mover_name="hard_design"/>
			<Add mover_name="hard_min"/>
			<Add mover_name="hard_design"/>
			<Add mover_name="RTmin"/>
			<Add mover_name="RTmin"/>
			<Add mover_name="hard_min"/>
		</ParsedProtocol>
                <LoopOver iterations="4" mover_name="design_block" name="iter4"/>
	</MOVERS>

	<FILTERS>
		<ScoreType confidence="0" name="stability_score_full" score_type="total_score" scorefxn="talaris_full" threshold="0"/>
		<ScoreType confidence="0" name="stability_without_pssm" score_type="total_score" scorefxn="tal_no_pssm" threshold="0"/>
		<ScoreType confidence="0" name="stability_pure" score_type="total_score" scorefxn="talaris_pure" threshold="0"/>
		<Rmsd confidence="0" name="rmsd"/>
		<Time name="timer"/>
	</FILTERS>


	<PROTOCOLS>
        <Add filter_name="timer"/>
		<Add mover_name="add_CA_cst"/>
		<Add mover_name="FSP"/>
		<Add mover_name="iter4"/>
		<Add filter_name="stability_score_full"/>
        <Add filter_name="stability_without_pssm"/>
        <Add filter_name="stability_pure"/>
		<Add filter_name="rmsd"/>
		<Add filter_name="timer"/>
	</PROTOCOLS>

</ROSETTASCRIPTS>

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flag文件

-ex1
-ex2
-use_input_sc
-extrachi_cutoff 5
-ignore_unrecognized_res
-use_occurrence_data
#-corrections::correct
#-corrections::score:no_his_his_pairE
-chemical:exclude_patches LowerDNA  UpperDNA Cterm_amidation SpecialRotamer VirtualBB ShoveBB VirtualDNAPhosphate VirtualNTerm CTermConnect sc_orbitals pro_hydroxylated_case1 pro_hydroxylated_case2 ser_phosphorylated thr_phosphorylated  tyr_phosphorylated tyr_sulfated lys_dimethylated lys_monomethylated  lys_trimethylated lys_acetylated glu_carboxylated cys_acetylated tyr_diiodinated N_acetylated C_methylamidated MethylatedProteinCterm
#-output_virtual
-linmem_ig 10
-ignore_zero_occupancy false

#-out:path:pdb pdbs/
#-out:path:score scores/
-restore_talaris_behavior true

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运行命令

结束会生成input_example_0001_0001.pdb和score.sc文件

/home/unbuntu/rosetta2019/main/source/bin/rosetta_scripts.default.linuxgccrelease -database /home/unbuntu/rosetta2019/main/database/ -in:file:s pdbs/input_example_0001.pdb -parser:protocol design.xml -parser:script_vars in_resfile=resfiles -parser:script_vars res_to_fix=1A,2A,3A,4A -parser:script_vars pdb_reference=pdbs/input_example_0001.pdb -parser:script_vars cst_full_path=example.cst -parser:script_vars cst_value=0.4 -parser:script_vars pssm_full_path=example.pssm @flags -overwrite
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运行filterscan

准备filterscan.xml文件

其中relax_mover=min_all删除，一直报错：没有min_all换了whole_min也报错

<ROSETTASCRIPTS>
    <SCOREFXNS> 
        <ScoreFunction name="scorefxn_full" weights="ref2015"> 
            <Reweight scoretype="coordinate_constraint" weight="0.1"/> 
            <Reweight scoretype="res_type_constraint" weight="0.1"/>
        </ScoreFunction>
    </SCOREFXNS>
    <RESIDUE_SELECTORS> 
       <Index name="ress_fix" resnums="%%res_to_fix%%"/>
    </RESIDUE_SELECTORS>
    <TASKOPERATIONS> 
        <InitializeFromCommandline name="init"/> 
        <DesignAround name="des_around" design_shell="0.1" resnums="%%current_res%%" repack_shell="8.0"/> 
        <SeqprofConsensus name="pssm_cutoff" filename="%%pssm_full_path%%" min_aa_probability="-2" probability_larger_than_current="0" convert_scores_to_probabilities="0" keep_native="1" debug="1" ignore_pose_profile_length_mismatch="0"/> 
        <OperateOnResidueSubset name="fix_res" selector="ress_fix"> 
            <PreventRepackingRLT/>
        </OperateOnResidueSubset>
        <OperateOnResidueSubset name="not_to_cst_sc"> 
            <Not selector="ress_fix"/> 
            <PreventRepackingRLT/> 
        </OperateOnResidueSubset>
    </TASKOPERATIONS>
    <FILTERS> 
        <ScoreType name="stability_score_full" scorefxn="scorefxn_full" score_type="total_score" threshold="0.0"/> 
        <Delta name="delta_score_full" filter="stability_score_full" upper="1" lower="0" range="0.5"/> 
        <FilterScan name="filter_scan" scorefxn="scorefxn_full" keep_native="1" task_operations="init,des_around,pssm_cutoff,fix_res" delta_filters="delta_score_full" delta="true" resfile_name="resfiles/res_%%current_res%%" report_all="1" delta_filter_thresholds="0.0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0" score_log_file="scores/res%%current_res%%_score_full.log" dump_pdb="1"/> 
    </FILTERS>
    <MOVERS> 
        <AtomCoordinateCstMover name="fix_res_sc_cst" coord_dev="0.5" bounded="false" sidechain="true" task_operations="not_to_cst_sc"/> 
        <ConstraintSetMover name="add_CA_cst" cst_file="%%cst_full_path%%"/> 
        <FavorSequenceProfile name="FSP" scaling="none" weight="1" pssm="%%pssm_full_path%%" scorefxns="scorefxn_full" /> 
        <MinMover name="whole_min" scorefxn="scorefxn_full" chi="1" bb="1" jump="0"/>
    </MOVERS>
    <PROTOCOLS> 
        <Add mover_name="add_CA_cst"/> 
        <Add mover_name="fix_res_sc_cst"/> 
        <Add mover_name="FSP"/> 
        <Add mover_name="whole_min"/> 
        <Add filter_name="filter_scan"/>
    </PROTOCOLS> 
    <OUTPUT scorefxn="scorefxn_full"/>
</ROSETTASCRIPTS>

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准备filterscan.flags

-use_input_sc 
-extrachi_cutoff 5 
-ignore_unrecognized_res
-chemical:exclude_patches LowerDNA UpperDNA Cterm_amidation SpecialRotamer VirtualBB ShoveBB VirtualDNAPhosphate VirtualNTerm CTermConnect sc_orbitals pro_hydroxylated_case1 pro_hydroxylated_case2 ser_phosphorylated thr_phosphorylated tyr_phosphorylated tyr_sulfated lys_dimethylated lys_monomethylated lys_trimethylated lys_acetylated glu_carboxylated cys_acetylated tyr_diiodinated N_acetylated C_methylamidated MethylatedProteinCterm
-linmem_ig 10 
-ignore_zero_occupancy false 
-s # path to structure file 
-out:path:pdb filterpdbs 
-out:path:score filterscores
-parser:protocol filterscan.xml 
-parser:script_vars current_res=1-73A # a position to mutational ddG for 
-parser:script_vars res_to_fix=1A,2A,3A,4A # comma separated list of positions 
-parser:script_vars cst_full_path=example.cst # path to Rosetta CST file of CA atoms 
-parser:script_vars pssm_full_path=example.pssm # path to pssm file

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/home/unbuntu/rosetta2019/main/source/bin/rosetta_scripts.default.linuxgccrelease -database /home/unbuntu/rosetta2019/main/database/ -in:file:s input_example_0001.pdb -parser:protocol filterscan.xml -parser:script_vars res_to_fix=1A,2A,3A,4A -parser:script_vars pdb_reference=input_example_0001.pdb -parser:script_vars res_to_restrict=10A,11A,12A,13A,14A,15A,16A,17A,18A -parser:script_vars cst_full_path=example.cst -parser:script_vars cst_value=0.4 -parser:script_vars pssm_full_path=example.pssm -parser:script_vars scores_path=scores/ -parser:script_vars resfiles_path=resfiles/ @flags -parser:script_vars current_res=${i} -overwrite
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创建两个文件夹filterscanpdbs和filterscanscores，生成的文件会放入对应文件夹。
运行命令：

/home/unbuntu/rosetta2019/main/source/bin/rosetta_scripts.default.linuxgccrelease -database /home/unbuntu/rosetta2019/main/database/ -in:file:s pdbs/input_example_0001.pdb -parser:protocol filterscan.xml -parser:script_vars res_to_fix=1A,2A,3A,4A -parser:script_vars pdb_reference=pdbs/input_example_0001.pdb -parser:script_vars res_to_restrict=10A,11A,12A,13A,14A,15A,16A,17A,18A -parser:script_vars cst_full_path=example.cst -parser:script_vars cst_value=0.4 -parser:script_vars pssm_full_path=example.pssm -parser:script_vars scores_path=scores/ -parser:script_vars resfiles_path=resfiles/ @filterscan.flags -parser:script_vars current_res=1-73A -overwrite
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其中current_res=1-73A参数，不确定是不是填序列长度。
current_res=${i}
i - the residue number (this script is for a single residue. you need to run it for every residue)