data_18357 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; The solution structure of the monomeric Acanthaporin ; _BMRB_accession_number 18357 _BMRB_flat_file_name bmr18357.str _Entry_type original _Submission_date 2012-03-28 _Accession_date 2012-03-28 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details . loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Michalek Matthias . . 2 Soennichsen Frank D. . 3 Wechselberger Rainer . . 4 Dingley Andrew J. . 5 Wienk Hans . . 6 Simanski Maren . . 7 Herbst Rosa . . 8 Lorenzen Inken . . 9 Marciano-Cabral Francine . . 10 Gelhaus Christoph . . 11 Groetzinger Joachim . . 12 Leippe Matthias . . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 247 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2012-05-01 original author . stop_ loop_ _Related_BMRB_accession_number _Relationship 18358 'dimeric Acanthaporin' stop_ _Original_release_date 2012-05-01 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title 'The solution structure of the monomeric Acanthaporin' _Citation_status submitted _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID ? loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Michalek Matthias . . 2 Soennichsen Frank D. . 3 Wechselberger Rainer . . 4 Dingley Andrew J. . 5 Wienk Hans . . 6 Simanski Maren . . 7 Herbst Rosa . . 8 Lorenzen Inken . . 9 Marciano-Cabral Francine . . 10 Gelhaus Christoph . . 11 Groetzinger Joachim . . 12 Leippe Matthias . . stop_ _Journal_abbreviation 'Not known' _Journal_volume . _Journal_issue . _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first . _Page_last . _Year . _Details . save_ ################################## # Molecular system description # ################################## save_assembly _Saveframe_category molecular_system _Mol_system_name Acanthaporin _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label Acanthaporin $acanthaporin stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state ? _System_paramagnetic no _System_thiol_state . _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_acanthaporin _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common acanthaporin _Molecular_mass 6001.235 _Mol_thiol_state 'all disulfide bound' _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 61 _Mol_residue_sequence ; AMGKCSVLKKVACAAAIAGA VAACGGIDLPCVLAALKAAE GCASCFCEDHCHGVCKDLHL C ; loop_ _Residue_seq_code _Residue_label 1 ALA 2 MET 3 GLY 4 LYS 5 CYS 6 SER 7 VAL 8 LEU 9 LYS 10 LYS 11 VAL 12 ALA 13 CYS 14 ALA 15 ALA 16 ALA 17 ILE 18 ALA 19 GLY 20 ALA 21 VAL 22 ALA 23 ALA 24 CYS 25 GLY 26 GLY 27 ILE 28 ASP 29 LEU 30 PRO 31 CYS 32 VAL 33 LEU 34 ALA 35 ALA 36 LEU 37 LYS 38 ALA 39 ALA 40 GLU 41 GLY 42 CYS 43 ALA 44 SER 45 CYS 46 PHE 47 CYS 48 GLU 49 ASP 50 HIS 51 CYS 52 HIS 53 GLY 54 VAL 55 CYS 56 LYS 57 ASP 58 LEU 59 HIS 60 LEU 61 CYS stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date 2014-03-04 loop_ _Database_name _Database_accession_code _Database_entry_mol_name _Sequence_query_to_submitted_percentage _Sequence_subject_length _Sequence_identity _Sequence_positive _Sequence_homology_expectation_value BMRB 16727 culbertcidin 100.00 61 100.00 100.00 6.78e-31 BMRB 16819 culbertcidin 100.00 122 100.00 100.00 1.71e-17 BMRB 18358 acanthaporin_dimer 100.00 61 100.00 100.00 6.78e-31 PDB 2LRD "The Solution Structure Of The Monomeric Acanthaporin" 100.00 61 100.00 100.00 6.78e-31 PDB 2LRE "The Solution Structure Of The Dimeric Acanthaporin" 100.00 61 100.00 100.00 6.78e-31 GB AEC33273 "acanthaporin precursor [Acanthamoeba culbertsoni]" 98.36 160 98.33 98.33 1.40e-29 stop_ save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $acanthaporin 'Acanthamoeba culbertsoni' 43142 Eukaryota . Acanthamoeba culbertsoni stop_ save_ ######################### # Experimental source # ######################### save_experimental_source _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_name $acanthaporin 'recombinant technology' . Escherichia coli . pET32a stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details '50 mM sodium phosphate buffer' loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $acanthaporin 1.0 mM '[U-100% 15N]' D2O 7 % [U-2H] 'sodium phosphate buffer' 50 mM 'natural abundance' 'sodium azide' 0.001 % 'natural abundance' H2O 93 % 'natural abundance' stop_ save_ ############################ # Computer software used # ############################ save_NMRPipe _Saveframe_category software _Name NMRPipe _Version . loop_ _Vendor _Address _Electronic_address 'Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax' . . stop_ loop_ _Task processing stop_ _Details . save_ save_NMRView _Saveframe_category software _Name NMRView _Version . loop_ _Vendor _Address _Electronic_address 'Johnson, One Moon Scientific' . . stop_ loop_ _Task 'chemical shift assignment' 'data analysis' 'peak picking' stop_ _Details . save_ save_CYANA _Saveframe_category software _Name CYANA _Version . loop_ _Vendor _Address _Electronic_address 'Guntert, Mumenthaler and Wuthrich' . . stop_ loop_ _Task 'structure solution' stop_ _Details . save_ save_X-PLOR_NIH _Saveframe_category software _Name 'X-PLOR NIH' _Version . loop_ _Vendor _Address _Electronic_address 'Schwieters, Kuszewski, Tjandra and Clore' . . stop_ loop_ _Task refinement stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model Avance _Field_strength 750 _Details 'Bruker DMX' save_ save_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model Avance _Field_strength 600 _Details 'Bruker DMX' save_ ############################# # NMR applied experiments # ############################# save_2D_1H-15N_HSQC_1 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-15N HSQC' _Sample_label $sample_1 save_ save_2D_1H-1H_TOCSY_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H TOCSY' _Sample_label $sample_1 save_ save_2D_1H-1H_NOESY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H NOESY' _Sample_label $sample_1 save_ save_3D_1H-15N_TOCSY_4 _Saveframe_category NMR_applied_experiment _Experiment_name '3D 1H-15N TOCSY' _Sample_label $sample_1 save_ save_3D_1H-15N_NOESY_5 _Saveframe_category NMR_applied_experiment _Experiment_name '3D 1H-15N NOESY' _Sample_label $sample_1 save_ ####################### # Sample conditions # ####################### save_sample_conditions_1 _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 0.05 . M pH 5.4 . pH pressure 1 . atm temperature 298 . K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chemical_shift_reference_1 _Saveframe_category chemical_shift_reference _Details . loop_ _Mol_common_name _Atom_type _Atom_isotope_number _Atom_group _Chem_shift_units _Chem_shift_value _Reference_method _Reference_type _External_reference_sample_geometry _External_reference_location _External_reference_axis _Indirect_shift_ratio water H 1 protons ppm 0 internal direct . . . 1.0 '[15N] ammonium chloride' N 15 nitrogen ppm 0 internal direct . . . 1.0 stop_ save_ ################################### # Assigned chemical shift lists # ################################### ################################################################### # Chemical Shift Ambiguity Index Value Definitions # # # # The values other than 1 are used for those atoms with different # # chemical shifts that cannot be assigned to stereospecific atoms # # or to specific residues or chains. # # # # Index Value Definition # # # # 1 Unique (including isolated methyl protons, # # geminal atoms, and geminal methyl # # groups with identical chemical shifts) # # (e.g. ILE HD11, HD12, HD13 protons) # # 2 Ambiguity of geminal atoms or geminal methyl # # proton groups (e.g. ASP HB2 and HB3 # # protons, LEU CD1 and CD2 carbons, or # # LEU HD11, HD12, HD13 and HD21, HD22, # # HD23 methyl protons) # # 3 Aromatic atoms on opposite sides of # # symmetrical rings (e.g. TYR HE1 and HE2 # # protons) # # 4 Intraresidue ambiguities (e.g. LYS HG and # # HD protons or TRP HZ2 and HZ3 protons) # # 5 Interresidue ambiguities (LYS 12 vs. LYS 27) # # 6 Intermolecular ambiguities (e.g. ASP 31 CA # # in monomer 1 and ASP 31 CA in monomer 2 # # of an asymmetrical homodimer, duplex # # DNA assignments, or other assignments # # that may apply to atoms in one or more # # molecule in the molecular assembly) # # 9 Ambiguous, specific ambiguity not defined # # # ################################################################### save_assigned_chem_shift_list_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Experiment_label '2D 1H-1H NOESY' '3D 1H-15N NOESY' stop_ loop_ _Sample_label $sample_1 stop_ _Sample_conditions_label $sample_conditions_1 _Chem_shift_reference_set_label $chemical_shift_reference_1 _Mol_system_component_name Acanthaporin _Text_data_format . _Text_data . loop_ _Atom_shift_assign_ID _Residue_author_seq_code _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code 1 2 2 MET H H 8.721 . 1 2 2 2 MET HA H 4.779 . 1 3 3 3 GLY H H 8.466 . 1 4 3 3 GLY HA2 H 4.001 . 2 5 3 3 GLY HA3 H 3.914 . 2 6 4 4 LYS H H 8.312 . 1 7 4 4 LYS HA H 4.319 . 1 8 4 4 LYS HB2 H 1.870 . 2 9 4 4 LYS HB3 H 1.663 . 2 10 4 4 LYS HG2 H 1.416 . 2 11 5 5 CYS H H 8.745 . 1 12 5 5 CYS HA H 4.582 . 1 13 5 5 CYS HB2 H 3.428 . 2 14 5 5 CYS HB3 H 2.589 . 2 15 6 6 SER H H 8.457 . 1 16 6 6 SER HA H 4.505 . 1 17 6 6 SER HB2 H 4.371 . 2 18 7 7 VAL H H 8.591 . 1 19 7 7 VAL HA H 3.740 . 1 20 7 7 VAL HB H 2.093 . 1 21 7 7 VAL HG1 H 1.097 . 4 22 7 7 VAL HG2 H 1.003 . 4 23 8 8 LEU H H 7.751 . 1 24 8 8 LEU HA H 4.140 . 1 25 8 8 LEU HB2 H 1.706 . 2 26 8 8 LEU HG H 1.503 . 1 27 8 8 LEU HD1 H 0.945 . 4 28 8 8 LEU HD2 H 0.889 . 4 29 9 9 LYS H H 7.834 . 1 30 9 9 LYS HA H 4.104 . 1 31 9 9 LYS HB2 H 2.176 . 2 32 9 9 LYS HB3 H 1.883 . 2 33 9 9 LYS HG2 H 1.687 . 2 34 10 10 LYS H H 8.602 . 1 35 10 10 LYS HA H 3.680 . 1 36 10 10 LYS HB2 H 2.021 . 2 37 11 11 VAL H H 7.550 . 1 38 11 11 VAL HA H 3.767 . 1 39 11 11 VAL HB H 2.162 . 1 40 11 11 VAL HG1 H 1.129 . 4 41 11 11 VAL HG2 H 1.002 . 4 42 12 12 ALA H H 7.911 . 1 43 12 12 ALA HA H 4.267 . 1 44 12 12 ALA HB H 1.600 . 1 45 13 13 CYS H H 8.379 . 1 46 13 13 CYS HA H 4.621 . 1 47 13 13 CYS HB2 H 3.044 . 2 48 13 13 CYS HB3 H 2.988 . 2 49 14 14 ALA H H 7.595 . 1 50 14 14 ALA HA H 4.043 . 1 51 14 14 ALA HB H 1.597 . 1 52 15 15 ALA H H 8.756 . 1 53 15 15 ALA HA H 4.235 . 1 54 15 15 ALA HB H 1.474 . 1 55 16 16 ALA H H 8.587 . 1 56 16 16 ALA HA H 4.100 . 1 57 16 16 ALA HB H 1.415 . 1 58 17 17 ILE H H 8.505 . 1 59 17 17 ILE HA H 4.196 . 1 60 17 17 ILE HB H 1.873 . 1 61 17 17 ILE HG12 H 1.076 . 9 62 17 17 ILE HG2 H 0.985 . 4 63 17 17 ILE HD1 H 0.832 . 1 64 18 18 ALA H H 8.304 . 1 65 18 18 ALA HA H 4.082 . 1 66 18 18 ALA HB H 1.493 . 1 67 19 19 GLY H H 8.402 . 1 68 19 19 GLY HA2 H 3.882 . 2 69 20 20 ALA H H 7.985 . 1 70 20 20 ALA HA H 4.091 . 1 71 20 20 ALA HB H 1.390 . 1 72 21 21 VAL H H 8.456 . 1 73 21 21 VAL HA H 3.417 . 1 74 21 21 VAL HB H 2.010 . 1 75 21 21 VAL HG1 H 0.945 . 4 76 21 21 VAL HG2 H 0.748 . 4 77 22 22 ALA H H 7.878 . 1 78 22 22 ALA HA H 4.107 . 1 79 22 22 ALA HB H 1.492 . 1 80 23 23 ALA H H 8.032 . 1 81 23 23 ALA HA H 4.120 . 1 82 23 23 ALA HB H 1.493 . 1 83 24 24 CYS H H 8.032 . 1 84 24 24 CYS HA H 4.497 . 1 85 24 24 CYS HB2 H 3.294 . 2 86 24 24 CYS HB3 H 3.027 . 2 87 25 25 GLY H H 7.977 . 1 88 25 25 GLY HA2 H 4.106 . 2 89 25 25 GLY HA3 H 3.852 . 2 90 26 26 GLY H H 8.098 . 1 91 26 26 GLY HA2 H 4.328 . 2 92 26 26 GLY HA3 H 3.646 . 2 93 27 27 ILE H H 8.317 . 1 94 27 27 ILE HA H 3.847 . 1 95 27 27 ILE HB H 2.073 . 1 96 27 27 ILE HG12 H 1.468 . 9 97 27 27 ILE HG13 H 1.294 . 9 98 27 27 ILE HG2 H 0.775 . 4 99 27 27 ILE HD1 H 0.716 . 1 100 28 28 ASP H H 7.216 . 1 101 28 28 ASP HA H 4.848 . 1 102 28 28 ASP HB2 H 2.642 . 2 103 28 28 ASP HB3 H 2.589 . 2 104 29 29 LEU H H 8.845 . 1 105 29 29 LEU HA H 4.034 . 1 106 29 29 LEU HB2 H 2.095 . 2 107 29 29 LEU HB3 H 2.005 . 2 108 29 29 LEU HG H 1.610 . 1 109 29 29 LEU HD1 H 0.984 . 4 110 30 30 PRO HA H 4.158 . 1 111 30 30 PRO HB2 H 2.248 . 2 112 30 30 PRO HG2 H 1.941 . 2 113 30 30 PRO HD2 H 3.758 . 2 114 31 31 CYS H H 7.310 . 1 115 31 31 CYS HA H 4.172 . 1 116 31 31 CYS HB2 H 3.126 . 2 117 31 31 CYS HB3 H 3.078 . 2 118 32 32 VAL H H 8.591 . 1 119 32 32 VAL HA H 3.208 . 1 120 32 32 VAL HB H 1.531 . 1 121 32 32 VAL HG1 H 0.717 . 4 122 32 32 VAL HG2 H -0.329 . 4 123 33 33 LEU H H 8.447 . 1 124 33 33 LEU HA H 3.707 . 1 125 33 33 LEU HB2 H 1.727 . 2 126 33 33 LEU HG H 1.330 . 1 127 33 33 LEU HD1 H 0.923 . 4 128 33 33 LEU HD2 H 0.806 . 4 129 34 34 ALA H H 7.540 . 1 130 34 34 ALA HA H 4.101 . 1 131 34 34 ALA HB H 1.486 . 1 132 35 35 ALA H H 7.796 . 1 133 35 35 ALA HA H 4.198 . 1 134 35 35 ALA HB H 1.502 . 1 135 36 36 LEU H H 7.850 . 1 136 36 36 LEU HA H 4.270 . 1 137 36 36 LEU HB2 H 1.740 . 2 138 36 36 LEU HB3 H 1.651 . 2 139 36 36 LEU HG H 1.562 . 1 140 36 36 LEU HD1 H 0.567 . 4 141 36 36 LEU HD2 H 0.425 . 4 142 37 37 LYS H H 7.336 . 1 143 37 37 LYS HA H 4.008 . 1 144 37 37 LYS HB2 H 1.934 . 2 145 37 37 LYS HB3 H 1.859 . 2 146 37 37 LYS HG2 H 1.596 . 2 147 38 38 ALA H H 7.863 . 1 148 38 38 ALA HA H 4.359 . 1 149 38 38 ALA HB H 1.414 . 1 150 39 39 ALA H H 7.862 . 1 151 39 39 ALA HA H 4.625 . 1 152 39 39 ALA HB H 1.228 . 1 153 40 40 GLU H H 8.105 . 1 154 40 40 GLU HA H 4.030 . 1 155 40 40 GLU HB2 H 2.030 . 2 156 40 40 GLU HG2 H 2.360 . 2 157 41 41 GLY H H 8.868 . 1 158 41 41 GLY HA2 H 4.177 . 2 159 41 41 GLY HA3 H 4.024 . 2 160 42 42 CYS H H 8.402 . 1 161 42 42 CYS HA H 4.827 . 1 162 42 42 CYS HB2 H 3.021 . 2 163 42 42 CYS HB3 H 2.950 . 2 164 43 43 ALA H H 9.090 . 1 165 43 43 ALA HA H 4.008 . 1 166 43 43 ALA HB H 1.560 . 1 167 44 44 SER H H 8.421 . 1 168 44 44 SER HA H 4.320 . 1 169 44 44 SER HB2 H 3.920 . 2 170 45 45 CYS H H 7.166 . 1 171 45 45 CYS HA H 4.335 . 1 172 45 45 CYS HB2 H 3.155 . 2 173 46 46 PHE H H 8.084 . 1 174 46 46 PHE HA H 4.610 . 1 175 46 46 PHE HB2 H 2.988 . 2 176 46 46 PHE HD1 H 6.643 . 3 177 46 46 PHE HE1 H 7.215 . 3 178 46 46 PHE HZ H 6.948 . 1 179 47 47 CYS H H 8.944 . 1 180 47 47 CYS HA H 4.213 . 1 181 47 47 CYS HB2 H 2.819 . 2 182 47 47 CYS HB3 H 2.602 . 2 183 48 48 GLU H H 7.982 . 1 184 48 48 GLU HA H 3.988 . 1 185 48 48 GLU HB2 H 2.138 . 2 186 48 48 GLU HG2 H 2.491 . 2 187 49 49 ASP H H 6.826 . 1 188 49 49 ASP HA H 4.627 . 1 189 49 49 ASP HB2 H 2.328 . 2 190 49 49 ASP HB3 H 2.249 . 2 191 50 50 HIS H H 7.297 . 1 192 50 50 HIS HA H 4.636 . 1 193 50 50 HIS HB2 H 2.865 . 2 194 50 50 HIS HB3 H 2.275 . 2 195 50 50 HIS HD1 H 6.549 . 3 196 50 50 HIS HE1 H 8.635 . 3 197 51 51 CYS H H 8.394 . 1 198 51 51 CYS HA H 4.509 . 1 199 51 51 CYS HB2 H 2.912 . 2 200 51 51 CYS HB3 H 2.888 . 2 201 52 52 HIS H H 8.321 . 1 202 52 52 HIS HA H 4.671 . 1 203 52 52 HIS HB2 H 3.422 . 2 204 52 52 HIS HB3 H 3.157 . 2 205 52 52 HIS HD1 H 7.086 . 3 206 52 52 HIS HE1 H 8.689 . 3 207 53 53 GLY H H 9.149 . 1 208 53 53 GLY HA2 H 4.077 . 2 209 53 53 GLY HA3 H 3.929 . 2 210 54 54 VAL H H 8.993 . 1 211 54 54 VAL HA H 4.053 . 1 212 54 54 VAL HB H 2.193 . 1 213 54 54 VAL HG1 H 1.055 . 4 214 55 55 CYS H H 7.249 . 1 215 55 55 CYS HA H 4.237 . 1 216 55 55 CYS HB2 H 3.400 . 2 217 55 55 CYS HB3 H 3.066 . 2 218 56 56 LYS H H 6.535 . 1 219 56 56 LYS HA H 4.108 . 1 220 56 56 LYS HB2 H 1.799 . 2 221 56 56 LYS HB3 H 1.390 . 2 222 56 56 LYS HG2 H 1.267 . 2 223 57 57 ASP H H 8.435 . 1 224 57 57 ASP HA H 4.153 . 1 225 57 57 ASP HB2 H 2.680 . 2 226 57 57 ASP HB3 H 2.553 . 2 227 58 58 LEU H H 7.690 . 1 228 58 58 LEU HA H 4.239 . 1 229 58 58 LEU HB2 H 1.797 . 2 230 58 58 LEU HB3 H 1.713 . 2 231 58 58 LEU HG H 1.612 . 1 232 58 58 LEU HD1 H 0.806 . 4 233 59 59 HIS H H 7.947 . 1 234 59 59 HIS HA H 4.392 . 1 235 59 59 HIS HB2 H 3.573 . 2 236 59 59 HIS HB3 H 3.423 . 2 237 59 59 HIS HD1 H 7.281 . 3 238 60 60 LEU H H 7.780 . 1 239 60 60 LEU HA H 4.348 . 1 240 60 60 LEU HB2 H 2.068 . 2 241 60 60 LEU HG H 1.294 . 1 242 60 60 LEU HD1 H 0.974 . 4 243 60 60 LEU HD2 H 0.818 . 4 244 61 61 CYS H H 7.654 . 1 245 61 61 CYS HA H 4.267 . 1 246 61 61 CYS HB2 H 3.118 . 2 247 61 61 CYS HB3 H 3.033 . 2 stop_ save_