data_27075 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; 1H and 15N Chemical Shift Assignments for phosphorylated S129 alpha-synuclein ; _BMRB_accession_number 27075 _BMRB_flat_file_name bmr27075.str _Entry_type original _Submission_date 2017-04-18 _Accession_date 2017-04-18 _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 'El Turk' Farah . . 2 'De Genst' Erwin . . 3 Guilliams Tim . . 4 Fauvet Bruno . . 5 Hejjaoui Mirva . . 6 Vendruscolo Michele . . 7 Lashuel Hilal A. . 8 Dobson Christopher M. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 126 "15N chemical shifts" 126 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2018-07-20 update BMRB 'update entry citation' 2018-05-22 original author 'original release' stop_ loop_ _Related_BMRB_accession_number _Relationship 27074 'wild-type alpha-synuclein' 27076 'Y133F/Y136F mutant alpha-synuclein' 27077 'phosphorylated Y125 Y133F/Y136F mutant alpha-synuclein' stop_ _Original_release_date 2017-04-18 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title ; Exploring the role of post-translational modifications in regulating alpha-synuclein interactions by studying the effects of phosphorylation on nanobody binding ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 29603451 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 'El Turk' Farah . . 2 'De Genst' Erwin . . 3 Guilliams Tim . . 4 Fauvet Bruno . . 5 Hejjaoui Mirva . . 6 'Di Trani' Justin . . 7 Chiki Anass . . 8 Mittermaier Anthony . . 9 Vendruscolo Michele . . 10 Lashuel Hilal A. . 11 Dobson Christopher M. . stop_ _Journal_abbreviation 'Protein Sci.' _Journal_name_full 'Protein science : a publication of the Protein Society' _Journal_volume 27 _Journal_issue 7 _Journal_ISSN 1469-896X _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first 1262 _Page_last 1274 _Year 2018 _Details . save_ ################################## # Molecular system description # ################################## save_assembly_1 _Saveframe_category molecular_system _Mol_system_name 'pS129 alpha-synuclein' _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label 'pS129 alpha-synuclein' $pS129_alpha-synuclein stop_ _System_molecular_weight . _System_physical_state 'intrinsically disordered' _System_oligomer_state ? _System_paramagnetic no _System_thiol_state . _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_pS129_alpha-synuclein _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common pS129_alpha-synuclein _Molecular_mass 14704 _Mol_thiol_state 'not present' _Details 'S129 is phosphorylated' ############################## # Polymer residue sequence # ############################## _Residue_count 140 _Mol_residue_sequence ; MDVFMKGLSKAKEGVVAAAE KTKQGVAEAAGKTKEGVLYV GSKTKEGVVHGVATVAEKTK EQVTNVGGAVVTGVTAVAQK TVEGAGSIAAATGFVKKDQL GKNEEGAPQEGILEDMPVDP DNEAYEMPXEEGYQDYEPEA ; loop_ _Residue_seq_code _Residue_label 1 MET 2 ASP 3 VAL 4 PHE 5 MET 6 LYS 7 GLY 8 LEU 9 SER 10 LYS 11 ALA 12 LYS 13 GLU 14 GLY 15 VAL 16 VAL 17 ALA 18 ALA 19 ALA 20 GLU 21 LYS 22 THR 23 LYS 24 GLN 25 GLY 26 VAL 27 ALA 28 GLU 29 ALA 30 ALA 31 GLY 32 LYS 33 THR 34 LYS 35 GLU 36 GLY 37 VAL 38 LEU 39 TYR 40 VAL 41 GLY 42 SER 43 LYS 44 THR 45 LYS 46 GLU 47 GLY 48 VAL 49 VAL 50 HIS 51 GLY 52 VAL 53 ALA 54 THR 55 VAL 56 ALA 57 GLU 58 LYS 59 THR 60 LYS 61 GLU 62 GLN 63 VAL 64 THR 65 ASN 66 VAL 67 GLY 68 GLY 69 ALA 70 VAL 71 VAL 72 THR 73 GLY 74 VAL 75 THR 76 ALA 77 VAL 78 ALA 79 GLN 80 LYS 81 THR 82 VAL 83 GLU 84 GLY 85 ALA 86 GLY 87 SER 88 ILE 89 ALA 90 ALA 91 ALA 92 THR 93 GLY 94 PHE 95 VAL 96 LYS 97 LYS 98 ASP 99 GLN 100 LEU 101 GLY 102 LYS 103 ASN 104 GLU 105 GLU 106 GLY 107 ALA 108 PRO 109 GLN 110 GLU 111 GLY 112 ILE 113 LEU 114 GLU 115 ASP 116 MET 117 PRO 118 VAL 119 ASP 120 PRO 121 ASP 122 ASN 123 GLU 124 ALA 125 TYR 126 GLU 127 MET 128 PRO 129 SEP 130 GLU 131 GLU 132 GLY 133 TYR 134 GLN 135 ASP 136 TYR 137 GLU 138 PRO 139 GLU 140 ALA stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date . save_ ###################### # Polymer residues # ###################### save_chem_comp_SEP _Saveframe_category polymer_residue _Mol_type 'L-PEPTIDE LINKING' _Name_common PHOSPHOSERINE _BMRB_code SEP _PDB_code SEP _Standard_residue_derivative . _Molecular_mass 185.072 _Mol_paramagnetic . _Details . loop_ _Atom_name _PDB_atom_name _Atom_type _Atom_chirality _Atom_charge _Atom_oxidation_number _Atom_unpaired_electrons N N N . 0 . ? CA CA C . 0 . ? CB CB C . 0 . ? OG OG O . 0 . ? C C C . 0 . ? O O O . 0 . ? OXT OXT O . 0 . ? P P P . 0 . ? O1P O1P O . 0 . ? O2P O2P O . 0 . ? O3P O3P O . 0 . ? H H H . 0 . ? H2 H2 H . 0 . ? HA HA H . 0 . ? HB2 HB2 H . 0 . ? HB3 HB3 H . 0 . ? HXT HXT H . 0 . ? HOP2 HOP2 H . 0 . ? HOP3 HOP3 H . 0 . ? stop_ loop_ _Bond_order _Bond_atom_one_atom_name _Bond_atom_two_atom_name _PDB_bond_atom_one_atom_name _PDB_bond_atom_two_atom_name SING N CA ? ? SING N H ? ? SING N H2 ? ? SING CA CB ? ? SING CA C ? ? SING CA HA ? ? SING CB OG ? ? SING CB HB2 ? ? SING CB HB3 ? ? SING OG P ? ? DOUB C O ? ? SING C OXT ? ? SING OXT HXT ? ? DOUB P O1P ? ? SING P O2P ? ? SING P O3P ? ? SING O2P HOP2 ? ? SING O3P HOP3 ? ? stop_ save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $pS129_alpha-synuclein 'E. coli' 562 Bacteria . Escherichia coli 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 $pS129_alpha-synuclein 'recombinant technology' . Escherichia coli . pET stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details ; Phosphorylated S129 alpha-synuclein 25 mM tris, 100 mM NaCl 25 uM DSS ; loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $pS129_alpha-synuclein 100 uM '[U-100% 13C; U-100% 15N]' NaCl 100 mM 'natural abundance' tris 25 mM 'natural abundance' stop_ save_ ############################ # Computer software used # ############################ save_NMRPipe _Saveframe_category software _Name NMRPipe _Version . loop_ _Vendor _Address _Electronic_address 'Delaglio, Zhengrong and Bax' . . stop_ loop_ _Task processing stop_ _Details . save_ save_SPARKY _Saveframe_category software _Name SPARKY _Version . loop_ _Vendor _Address _Electronic_address Goddard . . stop_ loop_ _Task 'chemical shift assignment' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Varian _Model INOVA _Field_strength 800 _Details ; Varian INOVA 800 MHz. 4 channels. Probe used was 13C-enhanced HCN cold probe with Z gradients. ; 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_ ####################### # Sample conditions # ####################### save_sample_conditions_1 _Saveframe_category sample_conditions _Details ; 25 mM tris, 100 mM NaCl pH 7.40 25 uM DSS ; loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 123 . mM pH 7.40 0.02 pH pressure 1 . atm temperature 273 . 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 DSS H 1 'methyl protons' ppm 4.931 internal indirect . . . 1 DSS N 15 'methyl protons' ppm 118.56 internal indirect . . . 0.101329118 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_ _Software_label $NMRPipe $SPARKY stop_ loop_ _Experiment_label '2D 1H-15N HSQC' 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 'pS129 alpha-synuclein' _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 3 3 VAL H H 8.293 . . 2 3 3 VAL N N 120.465 . . 3 4 4 PHE H H 8.400 . . 4 4 4 PHE N N 123.608 . . 5 5 5 MET H H 8.274 . . 6 5 5 MET N N 122.390 . . 7 6 6 LYS H H 8.334 . . 8 6 6 LYS N N 122.704 . . 9 7 7 GLY H H 8.471 . . 10 7 7 GLY N N 109.952 . . 11 8 8 LEU H H 8.116 . . 12 8 8 LEU N N 121.631 . . 13 9 9 SER H H 8.381 . . 14 9 9 SER N N 116.826 . . 15 10 10 LYS H H 8.428 . . 16 10 10 LYS N N 123.780 . . 17 11 11 ALA H H 8.367 . . 18 11 11 ALA N N 125.492 . . 19 12 12 LYS H H 8.405 . . 20 12 12 LYS N N 121.043 . . 21 13 13 GLU H H 8.506 . . 22 13 13 GLU N N 122.310 . . 23 14 14 GLY H H 8.526 . . 24 14 14 GLY N N 110.225 . . 25 15 15 VAL H H 8.044 . . 26 15 15 VAL N N 120.309 . . 27 17 17 ALA H H 8.519 . . 28 17 17 ALA N N 128.622 . . 29 18 18 ALA H H 8.384 . . 30 18 18 ALA N N 123.825 . . 31 19 19 ALA H H 8.344 . . 32 19 19 ALA N N 123.244 . . 33 20 20 GLU H H 8.396 . . 34 20 20 GLU N N 120.278 . . 35 21 21 LYS H H 8.403 . . 36 21 21 LYS N N 122.456 . . 37 22 22 THR H H 8.204 . . 38 22 22 THR N N 115.501 . . 39 23 23 LYS H H 8.417 . . 40 23 23 LYS N N 123.922 . . 41 24 24 GLN H H 8.497 . . 42 24 24 GLN N N 121.917 . . 43 25 25 GLY H H 8.546 . . 44 25 25 GLY N N 110.654 . . 45 26 26 VAL H H 8.081 . . 46 26 26 VAL N N 119.945 . . 47 27 27 ALA H H 8.486 . . 48 27 27 ALA N N 127.506 . . 49 28 28 GLU H H 8.468 . . 50 28 28 GLU N N 120.708 . . 51 29 29 ALA H H 8.365 . . 52 29 29 ALA N N 125.046 . . 53 30 30 ALA H H 8.300 . . 54 30 30 ALA N N 123.147 . . 55 31 31 GLY H H 8.372 . . 56 31 31 GLY N N 107.874 . . 57 32 32 LYS H H 8.166 . . 58 32 32 LYS N N 120.792 . . 59 33 33 THR H H 8.298 . . 60 33 33 THR N N 115.796 . . 61 34 34 LYS H H 8.547 . . 62 34 34 LYS N N 124.024 . . 63 36 36 GLY H H 8.496 . . 64 36 36 GLY N N 110.109 . . 65 37 37 VAL H H 7.973 . . 66 37 37 VAL N N 119.714 . . 67 38 38 LEU H H 8.352 . . 68 38 38 LEU N N 126.012 . . 69 39 39 TYR H H 8.341 . . 70 39 39 TYR N N 122.677 . . 71 40 40 VAL H H 8.145 . . 72 40 40 VAL N N 123.631 . . 73 41 41 GLY H H 8.095 . . 74 41 41 GLY N N 112.288 . . 75 44 44 THR H H 8.251 . . 76 44 44 THR N N 115.680 . . 77 45 45 LYS H H 8.506 . . 78 45 45 LYS N N 123.920 . . 79 48 48 VAL H H 7.975 . . 80 48 48 VAL N N 120.126 . . 81 49 49 VAL H H 8.373 . . 82 49 49 VAL N N 125.407 . . 83 51 51 GLY H H 8.463 . . 84 51 51 GLY N N 110.706 . . 85 52 52 VAL H H 8.097 . . 86 52 52 VAL N N 119.715 . . 87 53 53 ALA H H 8.559 . . 88 53 53 ALA N N 128.379 . . 89 54 54 THR H H 8.296 . . 90 54 54 THR N N 115.089 . . 91 55 55 VAL H H 8.317 . . 92 55 55 VAL N N 123.340 . . 93 56 56 ALA H H 8.485 . . 94 56 56 ALA N N 128.367 . . 95 57 57 GLU H H 8.445 . . 96 57 57 GLU N N 121.067 . . 97 58 58 LYS H H 8.504 . . 98 58 58 LYS N N 123.035 . . 99 59 59 THR H H 8.286 . . 100 59 59 THR N N 116.202 . . 101 60 60 LYS H H 8.465 . . 102 60 60 LYS N N 123.878 . . 103 61 61 GLU H H 8.513 . . 104 61 61 GLU N N 122.137 . . 105 62 62 GLN H H 8.493 . . 106 62 62 GLN N N 121.967 . . 107 63 63 VAL H H 8.357 . . 108 63 63 VAL N N 122.172 . . 109 64 64 THR H H 8.373 . . 110 64 64 THR N N 118.329 . . 111 65 65 ASN H H 8.587 . . 112 65 65 ASN N N 121.961 . . 113 66 66 VAL H H 8.313 . . 114 66 66 VAL N N 120.915 . . 115 67 67 GLY H H 8.624 . . 116 67 67 GLY N N 112.778 . . 117 68 68 GLY H H 8.293 . . 118 68 68 GLY N N 108.909 . . 119 69 69 ALA H H 8.228 . . 120 69 69 ALA N N 123.855 . . 121 70 70 VAL H H 8.291 . . 122 70 70 VAL N N 120.719 . . 123 71 71 VAL H H 8.471 . . 124 71 71 VAL N N 125.709 . . 125 72 72 THR H H 8.383 . . 126 72 72 THR N N 118.912 . . 127 73 73 GLY H H 8.504 . . 128 73 73 GLY N N 111.483 . . 129 74 74 VAL H H 8.151 . . 130 74 74 VAL N N 119.681 . . 131 75 75 THR H H 8.374 . . 132 75 75 THR N N 119.266 . . 133 76 76 ALA H H 8.445 . . 134 76 76 ALA N N 127.620 . . 135 77 77 VAL H H 8.223 . . 136 77 77 VAL N N 120.342 . . 137 78 78 ALA H H 8.492 . . 138 78 78 ALA N N 128.326 . . 139 79 79 GLN H H 8.467 . . 140 79 79 GLN N N 120.516 . . 141 80 80 LYS H H 8.507 . . 142 80 80 LYS N N 123.495 . . 143 81 81 THR H H 8.365 . . 144 81 81 THR N N 117.175 . . 145 82 82 VAL H H 8.380 . . 146 82 82 VAL N N 123.292 . . 147 83 83 GLU H H 8.644 . . 148 83 83 GLU N N 125.536 . . 149 84 84 GLY H H 8.589 . . 150 84 84 GLY N N 110.868 . . 151 85 85 ALA H H 8.326 . . 152 85 85 ALA N N 124.033 . . 153 86 86 GLY H H 8.562 . . 154 86 86 GLY N N 108.307 . . 155 87 87 SER H H 8.210 . . 156 87 87 SER N N 115.793 . . 157 88 88 ILE H H 8.261 . . 158 88 88 ILE N N 122.963 . . 159 89 89 ALA H H 8.420 . . 160 89 89 ALA N N 128.267 . . 161 90 90 ALA H H 8.285 . . 162 90 90 ALA N N 123.504 . . 163 91 91 ALA H H 8.362 . . 164 91 91 ALA N N 123.597 . . 165 92 92 THR H H 8.168 . . 166 92 92 THR N N 112.842 . . 167 93 93 GLY H H 8.372 . . 168 93 93 GLY N N 110.828 . . 169 94 94 PHE H H 8.153 . . 170 94 94 PHE N N 120.460 . . 171 95 95 VAL H H 8.120 . . 172 95 95 VAL N N 124.076 . . 173 96 96 LYS H H 8.463 . . 174 96 96 LYS N N 126.634 . . 175 97 97 LYS H H 8.541 . . 176 97 97 LYS N N 123.961 . . 177 98 98 ASP H H 8.469 . . 178 98 98 ASP N N 121.387 . . 179 99 99 GLN H H 8.410 . . 180 99 99 GLN N N 120.328 . . 181 100 100 LEU H H 8.367 . . 182 100 100 LEU N N 123.006 . . 183 101 101 GLY H H 8.546 . . 184 101 101 GLY N N 109.930 . . 185 102 102 LYS H H 8.274 . . 186 102 102 LYS N N 120.870 . . 187 103 103 ASN H H 8.688 . . 188 103 103 ASN N N 120.117 . . 189 104 104 GLU H H 8.541 . . 190 104 104 GLU N N 121.530 . . 191 105 105 GLU H H 8.532 . . 192 105 105 GLU N N 121.992 . . 193 106 106 GLY H H 8.500 . . 194 106 106 GLY N N 110.354 . . 195 107 107 ALA H H 8.188 . . 196 107 107 ALA N N 125.044 . . 197 109 109 GLN H H 8.662 . . 198 109 109 GLN N N 121.372 . . 199 110 110 GLU H H 8.598 . . 200 110 110 GLU N N 122.746 . . 201 111 111 GLY H H 8.553 . . 202 111 111 GLY N N 110.414 . . 203 112 112 ILE H H 8.069 . . 204 112 112 ILE N N 120.317 . . 205 113 113 LEU H H 8.484 . . 206 113 113 LEU N N 127.294 . . 207 114 114 GLU H H 8.496 . . 208 114 114 GLU N N 122.441 . . 209 115 115 ASP H H 8.430 . . 210 115 115 ASP N N 121.624 . . 211 116 116 MET H H 8.335 . . 212 116 116 MET N N 122.188 . . 213 118 118 VAL H H 8.382 . . 214 118 118 VAL N N 121.084 . . 215 119 119 ASP H H 8.605 . . 216 119 119 ASP N N 126.256 . . 217 121 121 ASP H H 8.448 . . 218 121 121 ASP N N 119.525 . . 219 122 122 ASN H H 8.187 . . 220 122 122 ASN N N 119.263 . . 221 123 123 GLU H H 8.433 . . 222 123 123 GLU N N 121.904 . . 223 124 124 ALA H H 8.296 . . 224 124 124 ALA N N 124.752 . . 225 125 125 TYR H H 8.128 . . 226 125 125 TYR N N 120.395 . . 227 126 126 GLU H H 8.226 . . 228 126 126 GLU N N 124.108 . . 229 127 127 MET H H 8.490 . . 230 127 127 MET N N 123.920 . . 231 129 129 SEP H H 9.098 . . 232 129 129 SEP N N 118.711 . . 233 130 130 GLU H H 8.609 . . 234 130 130 GLU N N 122.587 . . 235 131 131 GLU H H 8.539 . . 236 131 131 GLU N N 122.862 . . 237 132 132 GLY H H 8.478 . . 238 132 132 GLY N N 110.139 . . 239 133 133 TYR H H 8.160 . . 240 133 133 TYR N N 120.466 . . 241 134 134 GLN H H 8.273 . . 242 134 134 GLN N N 122.981 . . 243 135 135 ASP H H 8.303 . . 244 135 135 ASP N N 121.902 . . 245 136 136 TYR H H 8.117 . . 246 136 136 TYR N N 120.736 . . 247 137 137 GLU H H 8.331 . . 248 137 137 GLU N N 125.595 . . 249 139 139 GLU H H 8.590 . . 250 139 139 GLU N N 121.752 . . 251 140 140 ALA H H 8.076 . . 252 140 140 ALA N N 131.097 . . stop_ save_