data_50207 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; Purification of native CCL7 and its functional interaction with selected chemokine receptors ; _BMRB_accession_number 50207 _BMRB_flat_file_name bmr50207.str _Entry_type original _Submission_date 2020-03-07 _Accession_date 2020-03-07 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details ; Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C- isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and b-arrestins for selected chemokine receptors in cellular system. We compared cAMP response induced by histidine tagged CCL7 and native CCL7 and found that modification of the N-terminus of CCL7 compromises its functionality. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies. ; loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Goncharuk Marina V. . 2 Roy Debarati . . 3 Dubinnyi Maxim A. . 4 Nadezhdin Kirill D. . 5 Srivastava Ashish . . 6 Baidya Mithu . . 7 Dwivedi-Agnihotri Hemlata . . 8 Arseniev Alexander S. . 9 Shukla Arun K. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 445 "15N chemical shifts" 80 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2020-04-06 original BMRB . stop_ loop_ _Related_BMRB_accession_number _Relationship 4177 'This entry was used to transfer NMR assignment' 50208 'His6-tagged human chemokine CCL7' stop_ _Original_release_date 2020-03-20 save_ ############################# # Citation for this entry # ############################# save_citations_1 _Saveframe_category entry_citation _Citation_full . _Citation_title ; Purification of native CCL7 and its functional interaction with selected chemokine receptors ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 32145391 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Goncharuk Marina V. . 2 Roy Debarati . . 3 Dubinnyi Maxim A. . 4 Nadezhdin Kirill D. . 5 Srivastava Ashish . . 6 Baidya Mithu . . 7 Dwivedi-Agnihotri Hemlata . . 8 Arseniev Alexander S. . 9 Shukla Arun K. . stop_ _Journal_abbreviation 'Protein Expr. Purif.' _Journal_name_full 'Protein Expression and Purification' _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 2020 _Details . loop_ _Keyword Arrestins 'Atypical chemokine receptors' 'Biased agonism' Chemokine G-protein GPCR 'Isotope labeling; NMR spectroscopy' 'Recombinant protein' stop_ save_ ################################## # Molecular system description # ################################## save_assembly_1 _Saveframe_category molecular_system _Mol_system_name 'Native human chemokine CCL7' _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label natCCL7 $entity_1 stop_ _System_molecular_weight 8956 _System_physical_state native _System_oligomer_state ? _System_paramagnetic no _System_thiol_state . _Database_query_date . _Details 'Native human chemokine CCL7' save_ ######################## # Monomeric polymers # ######################## save_entity_1 _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common entity_1 _Molecular_mass . _Mol_thiol_state 'all disulfide bound' loop_ _Biological_function 'Chemotactic factor that attracts monocytes' stop_ _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 76 _Mol_residue_sequence ; QPVGINTSTTCCYRFINKKI PKQRLESYRRTTSSHCPREA VIFKTKLDKEICADPTQKWV QDFMKHLDKKTQTPKL ; loop_ _Residue_seq_code _Residue_label 1 GLN 2 PRO 3 VAL 4 GLY 5 ILE 6 ASN 7 THR 8 SER 9 THR 10 THR 11 CYS 12 CYS 13 TYR 14 ARG 15 PHE 16 ILE 17 ASN 18 LYS 19 LYS 20 ILE 21 PRO 22 LYS 23 GLN 24 ARG 25 LEU 26 GLU 27 SER 28 TYR 29 ARG 30 ARG 31 THR 32 THR 33 SER 34 SER 35 HIS 36 CYS 37 PRO 38 ARG 39 GLU 40 ALA 41 VAL 42 ILE 43 PHE 44 LYS 45 THR 46 LYS 47 LEU 48 ASP 49 LYS 50 GLU 51 ILE 52 CYS 53 ALA 54 ASP 55 PRO 56 THR 57 GLN 58 LYS 59 TRP 60 VAL 61 GLN 62 ASP 63 PHE 64 MET 65 LYS 66 HIS 67 LEU 68 ASP 69 LYS 70 LYS 71 THR 72 GLN 73 THR 74 PRO 75 LYS 76 LEU stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date . 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 SP P80098 . . . . . . stop_ save_ #################### # Natural source # #################### save_natural_source_1 _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $entity_1 Human 9606 Eukaryota Metazoa Homo sapiens stop_ save_ ######################### # Experimental source # ######################### save_experimental_source_1 _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_type _Vector_name $entity_1 'recombinant technology' . Escherichia coli BL21(DE3)pLysS plasmid pGEMEX-1(his6) stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details ; 303 oK pH 5.08 (KPi 6.12 titrated by d4-AcOD) 20mM KPi 0.4mM MES 7 mM NaCl ; loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $entity_1 0.45 mM '[U-100% 15N]' KPi 20 mM 'natural abundance' MES 0.4 mM 'natural abundance' NaCl 7 mM 'natural abundance' stop_ save_ ############################ # Computer software used # ############################ save_software_1 _Saveframe_category software _Name TOPSPIN _Version 3.0 loop_ _Task collection stop_ _Details . save_ save_software_2 _Saveframe_category software _Name CARA _Version 1.9.1.7 loop_ _Task 'chemical shift assignment' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model Avance _Field_strength 700 _Details 'Warm probe 5 mm PATXI' save_ save_NMR_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model 'Avance III' _Field_strength 800 _Details 'Cryoprobe 5 mm CPTCI' save_ ############################# # NMR applied experiments # ############################# save_1D_1H_1 _Saveframe_category NMR_applied_experiment _Experiment_name '1D 1H' _Sample_label $sample_1 save_ save_2D_1H-15N_HSQC_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-15N HSQC' _Sample_label $sample_1 save_ save_3D_1H-15N_TOCSY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '3D 1H-15N TOCSY' _Sample_label $sample_1 save_ save_3D_1H-15N_NOESY_4 _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' 0.007 . M pH 5.1 . pH pressure 1 . atm temperature 303 . K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chem_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 4.7 internal direct . . . 1 water N 15 protons ppm 4.7 internal indirect . . . 1 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_chemical_shifts_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Software_label $software_2 stop_ loop_ _Experiment_label '1D 1H' '2D 1H-15N HSQC' '3D 1H-15N TOCSY' '3D 1H-15N NOESY' stop_ loop_ _Sample_label $sample_1 stop_ _Sample_conditions_label $sample_conditions_1 _Chem_shift_reference_set_label $chem_shift_reference_1 _Mol_system_component_name natCCL7 _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 1 1 GLN HE21 H 7.497 0.020 1 2 1 1 GLN HE22 H 6.830 0.020 1 3 1 1 GLN NE2 N 112.186 0.3 1 4 3 3 VAL H H 8.323 0.020 1 5 3 3 VAL HA H 4.087 0.020 1 6 3 3 VAL HB H 2.061 0.020 1 7 3 3 VAL HG1 H 0.959 0.020 1 8 3 3 VAL HG2 H 0.959 0.020 1 9 3 3 VAL N N 120.777 0.3 1 10 4 4 GLY H H 8.428 0.020 1 11 4 4 GLY HA2 H 3.916 0.020 2 12 4 4 GLY HA3 H 3.998 0.020 2 13 4 4 GLY N N 112.338 0.3 1 14 5 5 ILE H H 7.939 0.020 1 15 5 5 ILE HA H 4.185 0.020 1 16 5 5 ILE HB H 1.837 0.020 1 17 5 5 ILE HG12 H 1.410 0.020 2 18 5 5 ILE HG13 H 1.160 0.020 2 19 5 5 ILE HG2 H 0.890 0.020 1 20 5 5 ILE HD1 H 0.830 0.020 1 21 5 5 ILE N N 119.629 0.3 1 22 6 6 ASN H H 8.550 0.020 1 23 6 6 ASN HA H 4.799 0.020 1 24 6 6 ASN HB2 H 2.859 0.020 2 25 6 6 ASN HB3 H 2.749 0.020 2 26 6 6 ASN HD21 H 7.561 0.020 1 27 6 6 ASN HD22 H 6.891 0.020 1 28 6 6 ASN N N 122.586 0.3 1 29 6 6 ASN ND2 N 112.815 0.3 1 30 7 7 THR H H 8.156 0.020 1 31 7 7 THR HA H 4.350 0.020 1 32 7 7 THR HB H 4.273 0.020 1 33 7 7 THR HG2 H 1.157 0.020 1 34 7 7 THR N N 114.510 0.3 1 35 8 8 SER H H 8.336 0.020 1 36 8 8 SER HA H 4.519 0.020 1 37 8 8 SER HB2 H 3.894 0.020 2 38 8 8 SER HB3 H 3.852 0.020 2 39 8 8 SER N N 117.869 0.3 1 40 9 9 THR H H 8.454 0.020 1 41 9 9 THR HA H 4.331 0.020 1 42 9 9 THR HB H 4.149 0.020 1 43 9 9 THR HG2 H 1.131 0.020 1 44 9 9 THR N N 117.029 0.3 1 45 10 10 THR H H 8.231 0.020 1 46 10 10 THR HA H 4.276 0.020 1 47 10 10 THR HB H 4.125 0.020 1 48 10 10 THR HG2 H 1.213 0.020 1 49 10 10 THR N N 118.269 0.3 1 50 11 11 CYS H H 8.087 0.020 1 51 11 11 CYS HA H 4.939 0.020 1 52 11 11 CYS HB2 H 2.755 0.020 2 53 11 11 CYS HB3 H 2.517 0.020 2 54 11 11 CYS N N 118.400 0.3 1 55 12 12 CYS H H 8.317 0.020 1 56 12 12 CYS HA H 4.592 0.020 1 57 12 12 CYS HB2 H 2.410 0.020 2 58 12 12 CYS HB3 H 2.663 0.020 2 59 12 12 CYS N N 118.645 0.3 1 60 14 14 ARG H H 7.403 0.020 1 61 14 14 ARG HA H 4.305 0.020 1 62 14 14 ARG HB2 H 1.750 0.020 2 63 14 14 ARG HB3 H 1.651 0.020 2 64 14 14 ARG HG2 H 1.478 0.020 2 65 14 14 ARG HG3 H 1.040 0.020 2 66 14 14 ARG HD2 H 3.175 0.020 1 67 14 14 ARG HD3 H 3.175 0.020 1 68 14 14 ARG HE H 7.180 0.020 1 69 14 14 ARG N N 118.805 0.3 1 70 15 15 PHE H H 8.546 0.020 1 71 15 15 PHE HA H 4.770 0.020 1 72 15 15 PHE HB2 H 2.900 0.020 2 73 15 15 PHE HB3 H 3.198 0.020 2 74 15 15 PHE HD1 H 7.030 0.020 1 75 15 15 PHE HD2 H 7.030 0.020 1 76 15 15 PHE HE1 H 7.200 0.020 1 77 15 15 PHE HE2 H 7.200 0.020 1 78 15 15 PHE HZ H 7.120 0.020 1 79 15 15 PHE N N 121.353 0.3 1 80 16 16 ILE H H 8.682 0.020 1 81 16 16 ILE HA H 4.395 0.020 1 82 16 16 ILE HB H 2.164 0.020 1 83 16 16 ILE HG12 H 1.620 0.020 2 84 16 16 ILE HG13 H 1.730 0.020 2 85 16 16 ILE HG2 H 1.216 0.020 1 86 16 16 ILE HD1 H 1.050 0.020 1 87 16 16 ILE N N 121.993 0.3 1 88 17 17 ASN H H 8.654 0.020 1 89 17 17 ASN HA H 5.022 0.020 1 90 17 17 ASN HB2 H 2.940 0.020 1 91 17 17 ASN HB3 H 2.940 0.020 1 92 17 17 ASN HD21 H 7.651 0.020 1 93 17 17 ASN HD22 H 6.954 0.020 1 94 17 17 ASN N N 123.127 0.3 1 95 17 17 ASN ND2 N 112.434 0.3 1 96 18 18 LYS H H 7.430 0.020 1 97 18 18 LYS HA H 4.236 0.020 1 98 18 18 LYS HB2 H 1.681 0.020 2 99 18 18 LYS HB3 H 1.550 0.020 2 100 18 18 LYS HG2 H 1.290 0.020 1 101 18 18 LYS HG3 H 1.290 0.020 1 102 18 18 LYS HD2 H 1.576 0.020 1 103 18 18 LYS HD3 H 1.576 0.020 1 104 18 18 LYS HE2 H 2.950 0.020 1 105 18 18 LYS HE3 H 2.950 0.020 1 106 18 18 LYS N N 119.854 0.3 1 107 19 19 LYS H H 7.705 0.020 1 108 19 19 LYS HA H 2.452 0.020 1 109 19 19 LYS HB2 H 1.006 0.020 2 110 19 19 LYS HB3 H 0.959 0.020 2 111 19 19 LYS HG2 H 0.700 0.020 2 112 19 19 LYS HG3 H 0.558 0.020 2 113 19 19 LYS HD2 H 1.323 0.020 1 114 19 19 LYS HD3 H 1.323 0.020 1 115 19 19 LYS HE2 H 2.731 0.020 1 116 19 19 LYS HE3 H 2.731 0.020 1 117 19 19 LYS N N 120.750 0.3 1 118 22 22 LYS H H 8.291 0.020 1 119 22 22 LYS HA H 3.280 0.020 1 120 22 22 LYS HB2 H 1.332 0.020 2 121 22 22 LYS HB3 H 1.222 0.020 2 122 22 22 LYS HG2 H 0.647 0.020 2 123 22 22 LYS HG3 H 0.834 0.020 2 124 22 22 LYS HD2 H 1.090 0.020 2 125 22 22 LYS HD3 H 1.172 0.020 2 126 22 22 LYS HE2 H 2.309 0.020 1 127 22 22 LYS HE3 H 2.309 0.020 1 128 22 22 LYS N N 124.477 0.3 1 129 23 23 GLN H H 8.608 0.020 1 130 23 23 GLN HA H 4.159 0.020 1 131 23 23 GLN HB2 H 2.036 0.020 1 132 23 23 GLN HB3 H 2.036 0.020 1 133 23 23 GLN HG2 H 2.430 0.020 2 134 23 23 GLN HG3 H 2.370 0.020 2 135 23 23 GLN HE21 H 7.556 0.020 1 136 23 23 GLN HE22 H 6.886 0.020 1 137 23 23 GLN N N 116.301 0.3 1 138 23 23 GLN NE2 N 111.200 0.3 1 139 24 24 ARG H H 7.782 0.020 1 140 24 24 ARG HA H 4.297 0.020 1 141 24 24 ARG HB2 H 2.043 0.020 2 142 24 24 ARG HB3 H 1.770 0.020 2 143 24 24 ARG HG2 H 1.674 0.020 1 144 24 24 ARG HG3 H 1.674 0.020 1 145 24 24 ARG HD2 H 3.130 0.020 2 146 24 24 ARG HD3 H 3.190 0.020 2 147 24 24 ARG HE H 7.171 0.020 1 148 24 24 ARG N N 116.254 0.3 1 149 24 24 ARG NE N 84.565 0.3 1 150 25 25 LEU H H 7.525 0.020 1 151 25 25 LEU HA H 4.278 0.020 1 152 25 25 LEU HB2 H 1.885 0.020 2 153 25 25 LEU HB3 H 1.260 0.020 2 154 25 25 LEU HG H 1.546 0.020 1 155 25 25 LEU HD1 H 0.234 0.020 2 156 25 25 LEU HD2 H 0.498 0.020 2 157 25 25 LEU N N 119.038 0.3 1 158 26 26 GLU H H 9.109 0.020 1 159 26 26 GLU HA H 4.605 0.020 1 160 26 26 GLU HB2 H 1.909 0.020 2 161 26 26 GLU HB3 H 1.715 0.020 2 162 26 26 GLU HG2 H 2.183 0.020 1 163 26 26 GLU HG3 H 2.183 0.020 1 164 26 26 GLU N N 120.373 0.3 1 165 27 27 SER H H 8.042 0.020 1 166 27 27 SER HA H 4.766 0.020 1 167 27 27 SER HB2 H 3.984 0.020 2 168 27 27 SER HB3 H 4.059 0.020 2 169 27 27 SER N N 109.150 0.3 1 170 28 28 TYR H H 8.609 0.020 1 171 28 28 TYR HA H 5.704 0.020 1 172 28 28 TYR HB2 H 2.377 0.020 2 173 28 28 TYR HB3 H 2.871 0.020 2 174 28 28 TYR HD1 H 6.770 0.020 1 175 28 28 TYR HD2 H 6.770 0.020 1 176 28 28 TYR HE1 H 6.860 0.020 1 177 28 28 TYR HE2 H 6.860 0.020 1 178 28 28 TYR N N 115.645 0.3 1 179 29 29 ARG H H 8.524 0.020 1 180 29 29 ARG HA H 4.551 0.020 1 181 29 29 ARG HB2 H 1.808 0.020 2 182 29 29 ARG HB3 H 1.670 0.020 2 183 29 29 ARG HG2 H 1.333 0.020 1 184 29 29 ARG HG3 H 1.333 0.020 1 185 29 29 ARG HD2 H 3.025 0.020 2 186 29 29 ARG HD3 H 3.151 0.020 2 187 29 29 ARG HE H 7.496 0.020 1 188 29 29 ARG N N 117.231 0.3 1 189 29 29 ARG NE N 85.375 0.3 1 190 30 30 ARG H H 8.849 0.020 1 191 30 30 ARG HA H 5.157 0.020 1 192 30 30 ARG HB2 H 1.702 0.020 2 193 30 30 ARG HB3 H 2.064 0.020 2 194 30 30 ARG HG2 H 1.775 0.020 2 195 30 30 ARG HG3 H 1.615 0.020 2 196 30 30 ARG HD2 H 3.235 0.020 1 197 30 30 ARG HD3 H 3.235 0.020 1 198 30 30 ARG HE H 7.406 0.020 1 199 30 30 ARG N N 121.064 0.3 1 200 30 30 ARG NE N 84.294 0.3 1 201 31 31 THR H H 8.399 0.020 1 202 31 31 THR HA H 4.590 0.020 1 203 31 31 THR HB H 4.557 0.020 1 204 31 31 THR HG2 H 1.217 0.020 1 205 31 31 THR N N 113.173 0.3 1 206 32 32 THR H H 8.293 0.020 1 207 32 32 THR HA H 4.488 0.020 1 208 32 32 THR HB H 4.295 0.020 1 209 32 32 THR HG2 H 1.184 0.020 1 210 32 32 THR N N 113.977 0.3 1 211 33 33 SER H H 8.426 0.020 1 212 33 33 SER HA H 4.474 0.020 1 213 33 33 SER HB2 H 3.990 0.020 2 214 33 33 SER HB3 H 3.880 0.020 2 215 33 33 SER N N 117.798 0.3 1 216 34 34 SER H H 8.472 0.020 1 217 34 34 SER HA H 4.301 0.020 1 218 34 34 SER HB2 H 3.853 0.020 2 219 34 34 SER HB3 H 3.821 0.020 2 220 34 34 SER N N 119.106 0.3 1 221 35 35 HIS H H 8.113 0.020 1 222 35 35 HIS HA H 4.720 0.020 1 223 35 35 HIS HB2 H 3.093 0.020 2 224 35 35 HIS HB3 H 3.327 0.020 2 225 35 35 HIS N N 116.791 0.3 1 226 36 36 CYS H H 7.681 0.020 1 227 36 36 CYS HA H 5.151 0.020 1 228 36 36 CYS HB2 H 2.661 0.020 2 229 36 36 CYS HB3 H 3.336 0.020 2 230 36 36 CYS N N 117.965 0.3 1 231 38 38 ARG H H 7.159 0.020 1 232 38 38 ARG HA H 4.527 0.020 1 233 38 38 ARG HB2 H 1.686 0.020 2 234 38 38 ARG HB3 H 1.866 0.020 2 235 38 38 ARG HG2 H 1.509 0.020 1 236 38 38 ARG HG3 H 1.509 0.020 1 237 38 38 ARG HD2 H 3.027 0.020 1 238 38 38 ARG HD3 H 3.027 0.020 1 239 38 38 ARG HE H 7.124 0.020 1 240 38 38 ARG N N 113.207 0.3 1 241 38 38 ARG NE N 84.099 0.3 1 242 39 39 GLU H H 8.609 0.020 1 243 39 39 GLU HA H 4.057 0.020 1 244 39 39 GLU HB2 H 1.918 0.020 1 245 39 39 GLU HB3 H 1.918 0.020 1 246 39 39 GLU HG2 H 2.290 0.020 2 247 39 39 GLU HG3 H 2.149 0.020 2 248 39 39 GLU N N 123.894 0.3 1 249 40 40 ALA H H 8.088 0.020 1 250 40 40 ALA HA H 4.943 0.020 1 251 40 40 ALA HB H 1.547 0.020 1 252 40 40 ALA N N 122.807 0.3 1 253 41 41 VAL H H 8.403 0.020 1 254 41 41 VAL HA H 4.434 0.020 1 255 41 41 VAL HB H 1.322 0.020 1 256 41 41 VAL HG1 H 0.583 0.020 2 257 41 41 VAL HG2 H 0.088 0.020 2 258 41 41 VAL N N 119.821 0.3 1 259 42 42 ILE H H 8.953 0.020 1 260 42 42 ILE HA H 4.909 0.020 1 261 42 42 ILE HB H 1.604 0.020 1 262 42 42 ILE HG12 H 1.410 0.020 2 263 42 42 ILE HG13 H 0.900 0.020 2 264 42 42 ILE HG2 H 0.720 0.020 1 265 42 42 ILE HD1 H 0.627 0.020 1 266 42 42 ILE N N 124.851 0.3 1 267 43 43 PHE H H 9.291 0.020 1 268 43 43 PHE HA H 5.284 0.020 1 269 43 43 PHE HB2 H 2.915 0.020 2 270 43 43 PHE HB3 H 3.100 0.020 2 271 43 43 PHE HD1 H 7.240 0.020 1 272 43 43 PHE HD2 H 7.240 0.020 1 273 43 43 PHE HE1 H 6.910 0.020 1 274 43 43 PHE HE2 H 6.910 0.020 1 275 43 43 PHE HZ H 7.260 0.020 1 276 43 43 PHE N N 126.981 0.3 1 277 44 44 LYS H H 8.946 0.020 1 278 44 44 LYS HA H 5.325 0.020 1 279 44 44 LYS HB2 H 1.965 0.020 2 280 44 44 LYS HB3 H 1.816 0.020 2 281 44 44 LYS HG2 H 1.400 0.020 1 282 44 44 LYS HG3 H 1.400 0.020 1 283 44 44 LYS HD2 H 1.640 0.020 1 284 44 44 LYS HD3 H 1.640 0.020 1 285 44 44 LYS HE2 H 2.950 0.020 1 286 44 44 LYS HE3 H 2.950 0.020 1 287 44 44 LYS N N 122.338 0.3 1 288 45 45 THR H H 9.020 0.020 1 289 45 45 THR HA H 5.177 0.020 1 290 45 45 THR HB H 4.727 0.020 1 291 45 45 THR HG2 H 1.184 0.020 1 292 45 45 THR N N 116.428 0.3 1 293 46 46 LYS H H 8.637 0.020 1 294 46 46 LYS HA H 4.174 0.020 1 295 46 46 LYS HB2 H 1.740 0.020 2 296 46 46 LYS HB3 H 1.976 0.020 2 297 46 46 LYS HG2 H 1.400 0.020 2 298 46 46 LYS HG3 H 1.500 0.020 2 299 46 46 LYS HD2 H 1.500 0.020 1 300 46 46 LYS HD3 H 1.500 0.020 1 301 46 46 LYS HE2 H 2.986 0.020 1 302 46 46 LYS HE3 H 2.986 0.020 1 303 46 46 LYS N N 119.603 0.3 1 304 47 47 LEU H H 7.704 0.020 1 305 47 47 LEU HA H 4.450 0.020 1 306 47 47 LEU HB2 H 1.783 0.020 2 307 47 47 LEU HB3 H 1.560 0.020 2 308 47 47 LEU HG H 1.590 0.020 1 309 47 47 LEU HD1 H 0.930 0.020 2 310 47 47 LEU HD2 H 0.870 0.020 2 311 47 47 LEU N N 117.670 0.3 1 312 48 48 ASP H H 8.014 0.020 1 313 48 48 ASP HA H 4.324 0.020 1 314 48 48 ASP HB2 H 2.990 0.020 2 315 48 48 ASP HB3 H 2.638 0.020 2 316 48 48 ASP N N 116.188 0.3 1 317 49 49 LYS H H 7.408 0.020 1 318 49 49 LYS HA H 4.550 0.020 1 319 49 49 LYS HB2 H 1.755 0.020 2 320 49 49 LYS HB3 H 1.835 0.020 2 321 49 49 LYS HG2 H 1.301 0.020 1 322 49 49 LYS HG3 H 1.301 0.020 1 323 49 49 LYS HD2 H 1.432 0.020 1 324 49 49 LYS HD3 H 1.432 0.020 1 325 49 49 LYS HE2 H 2.999 0.020 1 326 49 49 LYS HE3 H 2.999 0.020 1 327 49 49 LYS N N 117.181 0.3 1 328 50 50 GLU H H 8.435 0.020 1 329 50 50 GLU HA H 5.375 0.020 1 330 50 50 GLU HB2 H 1.960 0.020 2 331 50 50 GLU HB3 H 1.763 0.020 2 332 50 50 GLU HG2 H 2.000 0.020 2 333 50 50 GLU HG3 H 2.400 0.020 2 334 50 50 GLU N N 120.268 0.3 1 335 51 51 ILE H H 9.155 0.020 1 336 51 51 ILE HA H 4.478 0.020 1 337 51 51 ILE HB H 1.924 0.020 1 338 51 51 ILE HG12 H 1.201 0.020 2 339 51 51 ILE HG13 H 1.518 0.020 2 340 51 51 ILE HG2 H 1.109 0.020 1 341 51 51 ILE HD1 H 0.861 0.020 1 342 51 51 ILE N N 122.180 0.3 1 343 52 52 CYS H H 8.841 0.020 1 344 52 52 CYS HA H 5.034 0.020 1 345 52 52 CYS HB2 H 3.421 0.020 2 346 52 52 CYS HB3 H 2.789 0.020 2 347 52 52 CYS N N 125.976 0.3 1 348 53 53 ALA H H 9.831 0.020 1 349 53 53 ALA HA H 4.948 0.020 1 350 53 53 ALA HB H 1.302 0.020 1 351 53 53 ALA N N 127.206 0.3 1 352 54 54 ASP H H 8.396 0.020 1 353 54 54 ASP HA H 4.040 0.020 1 354 54 54 ASP HB2 H 2.568 0.020 2 355 54 54 ASP HB3 H 1.623 0.020 2 356 54 54 ASP N N 121.425 0.3 1 357 56 56 THR H H 8.230 0.020 1 358 56 56 THR HA H 4.018 0.020 1 359 56 56 THR HB H 4.163 0.020 1 360 56 56 THR HG2 H 1.167 0.020 1 361 56 56 THR N N 109.750 0.3 1 362 57 57 GLN H H 7.412 0.020 1 363 57 57 GLN HA H 4.170 0.020 1 364 57 57 GLN HB2 H 1.611 0.020 2 365 57 57 GLN HB3 H 1.826 0.020 2 366 57 57 GLN HG2 H 2.353 0.020 2 367 57 57 GLN HG3 H 2.207 0.020 2 368 57 57 GLN HE21 H 7.359 0.020 1 369 57 57 GLN HE22 H 6.963 0.020 1 370 57 57 GLN N N 120.688 0.3 1 371 57 57 GLN NE2 N 113.336 0.3 1 372 58 58 LYS H H 8.763 0.020 1 373 58 58 LYS HA H 3.753 0.020 1 374 58 58 LYS HB2 H 1.909 0.020 1 375 58 58 LYS HB3 H 1.909 0.020 1 376 58 58 LYS HG2 H 1.449 0.020 1 377 58 58 LYS HG3 H 1.449 0.020 1 378 58 58 LYS HD2 H 1.726 0.020 2 379 58 58 LYS HD3 H 1.618 0.020 2 380 58 58 LYS HE2 H 3.028 0.020 1 381 58 58 LYS HE3 H 3.028 0.020 1 382 58 58 LYS N N 126.026 0.3 1 383 59 59 TRP H H 8.271 0.020 1 384 59 59 TRP HA H 4.282 0.020 1 385 59 59 TRP HB2 H 3.050 0.020 2 386 59 59 TRP HB3 H 3.010 0.020 2 387 59 59 TRP HD1 H 7.580 0.020 1 388 59 59 TRP HE1 H 10.009 0.020 1 389 59 59 TRP HE3 H 6.350 0.020 1 390 59 59 TRP HZ2 H 7.380 0.020 1 391 59 59 TRP HZ3 H 6.510 0.020 1 392 59 59 TRP HH2 H 6.940 0.020 1 393 59 59 TRP N N 115.553 0.3 1 394 59 59 TRP NE1 N 129.477 0.3 1 395 60 60 VAL H H 5.691 0.020 1 396 60 60 VAL HA H 2.774 0.020 1 397 60 60 VAL HB H 1.766 0.020 1 398 60 60 VAL HG1 H -0.714 0.020 2 399 60 60 VAL HG2 H 0.420 0.020 2 400 60 60 VAL N N 122.626 0.3 1 401 61 61 GLN H H 7.194 0.020 1 402 61 61 GLN HA H 3.947 0.020 1 403 61 61 GLN HB2 H 2.140 0.020 2 404 61 61 GLN HB3 H 2.080 0.020 2 405 61 61 GLN HG2 H 2.368 0.020 2 406 61 61 GLN HG3 H 2.257 0.020 2 407 61 61 GLN HE21 H 6.604 0.020 1 408 61 61 GLN HE22 H 7.369 0.020 1 409 61 61 GLN N N 118.581 0.3 1 410 61 61 GLN NE2 N 110.462 0.3 1 411 62 62 ASP H H 8.636 0.020 1 412 62 62 ASP HA H 4.429 0.020 1 413 62 62 ASP HB2 H 2.870 0.020 2 414 62 62 ASP HB3 H 2.745 0.020 2 415 62 62 ASP N N 120.027 0.3 1 416 63 63 PHE H H 8.551 0.020 1 417 63 63 PHE HA H 4.550 0.020 1 418 63 63 PHE HB2 H 3.530 0.020 2 419 63 63 PHE HB3 H 2.900 0.020 2 420 63 63 PHE HD1 H 7.010 0.020 1 421 63 63 PHE HD2 H 7.010 0.020 1 422 63 63 PHE HE1 H 7.440 0.020 1 423 63 63 PHE HE2 H 7.440 0.020 1 424 63 63 PHE HZ H 7.380 0.020 1 425 63 63 PHE N N 122.392 0.3 1 426 64 64 MET H H 8.394 0.020 1 427 64 64 MET HA H 3.662 0.020 1 428 64 64 MET HB2 H 2.390 0.020 2 429 64 64 MET HB3 H 2.090 0.020 2 430 64 64 MET HG2 H 2.280 0.020 2 431 64 64 MET HG3 H 1.745 0.020 2 432 64 64 MET HE H 1.790 0.020 1 433 64 64 MET N N 119.622 0.3 1 434 65 65 LYS H H 7.620 0.020 1 435 65 65 LYS HA H 4.115 0.020 1 436 65 65 LYS HB2 H 1.963 0.020 2 437 65 65 LYS HB3 H 1.740 0.020 2 438 65 65 LYS HG2 H 1.509 0.020 2 439 65 65 LYS HG3 H 1.660 0.020 2 440 65 65 LYS HD2 H 1.700 0.020 1 441 65 65 LYS HD3 H 1.700 0.020 1 442 65 65 LYS HE2 H 2.994 0.020 1 443 65 65 LYS HE3 H 2.994 0.020 1 444 65 65 LYS N N 116.764 0.3 1 445 66 66 HIS H H 7.868 0.020 1 446 66 66 HIS HA H 4.393 0.020 1 447 66 66 HIS HB2 H 3.412 0.020 2 448 66 66 HIS HB3 H 3.240 0.020 2 449 66 66 HIS HD2 H 6.700 0.020 1 450 66 66 HIS HE1 H 7.220 0.020 1 451 66 66 HIS N N 117.573 0.3 1 452 67 67 LEU H H 8.122 0.020 1 453 67 67 LEU HA H 4.069 0.020 1 454 67 67 LEU HB2 H 2.220 0.020 2 455 67 67 LEU HB3 H 1.744 0.020 2 456 67 67 LEU HG H 2.010 0.020 1 457 67 67 LEU HD1 H 1.110 0.020 2 458 67 67 LEU HD2 H 0.818 0.020 2 459 67 67 LEU N N 119.648 0.3 1 460 68 68 ASP H H 8.604 0.020 1 461 68 68 ASP HA H 4.569 0.020 1 462 68 68 ASP HB2 H 2.866 0.020 2 463 68 68 ASP HB3 H 2.749 0.020 2 464 68 68 ASP N N 121.166 0.3 1 465 69 69 LYS H H 7.563 0.020 1 466 69 69 LYS HA H 4.191 0.020 1 467 69 69 LYS HB2 H 1.840 0.020 2 468 69 69 LYS HB3 H 1.890 0.020 2 469 69 69 LYS HG2 H 1.458 0.020 1 470 69 69 LYS HG3 H 1.458 0.020 1 471 69 69 LYS HD2 H 1.567 0.020 1 472 69 69 LYS HD3 H 1.567 0.020 1 473 69 69 LYS HE2 H 2.994 0.020 1 474 69 69 LYS HE3 H 2.994 0.020 1 475 69 69 LYS N N 118.926 0.3 1 476 70 70 LYS H H 7.867 0.020 1 477 70 70 LYS HA H 4.264 0.020 1 478 70 70 LYS HB2 H 1.890 0.020 2 479 70 70 LYS HB3 H 1.804 0.020 2 480 70 70 LYS HG2 H 1.442 0.020 1 481 70 70 LYS HG3 H 1.442 0.020 1 482 70 70 LYS HD2 H 1.667 0.020 1 483 70 70 LYS HD3 H 1.667 0.020 1 484 70 70 LYS HE2 H 3.005 0.020 1 485 70 70 LYS HE3 H 3.005 0.020 1 486 70 70 LYS N N 120.265 0.3 1 487 71 71 THR H H 7.953 0.020 1 488 71 71 THR HA H 4.350 0.020 1 489 71 71 THR HB H 4.280 0.020 1 490 71 71 THR HG2 H 1.240 0.020 1 491 71 71 THR N N 113.356 0.3 1 492 72 72 GLN H H 8.194 0.020 1 493 72 72 GLN HA H 4.408 0.020 1 494 72 72 GLN HB2 H 2.004 0.020 2 495 72 72 GLN HB3 H 2.120 0.020 2 496 72 72 GLN HG2 H 2.390 0.020 1 497 72 72 GLN HG3 H 2.390 0.020 1 498 72 72 GLN HE21 H 7.554 0.020 1 499 72 72 GLN HE22 H 6.882 0.020 1 500 72 72 GLN N N 122.337 0.3 1 501 72 72 GLN NE2 N 111.686 0.3 1 502 73 73 THR H H 8.190 0.020 1 503 73 73 THR HA H 4.565 0.020 1 504 73 73 THR HB H 4.141 0.020 1 505 73 73 THR HG2 H 1.247 0.020 1 506 73 73 THR N N 118.197 0.3 1 507 75 75 LYS H H 8.332 0.020 1 508 75 75 LYS HA H 4.301 0.020 1 509 75 75 LYS HB2 H 1.850 0.020 2 510 75 75 LYS HB3 H 1.750 0.020 2 511 75 75 LYS HG2 H 1.448 0.020 1 512 75 75 LYS HG3 H 1.448 0.020 1 513 75 75 LYS HD2 H 1.700 0.020 1 514 75 75 LYS HD3 H 1.700 0.020 1 515 75 75 LYS HE2 H 2.996 0.020 1 516 75 75 LYS HE3 H 2.996 0.020 1 517 75 75 LYS N N 122.409 0.3 1 518 76 76 LEU H H 7.868 0.020 1 519 76 76 LEU HA H 4.187 0.020 1 520 76 76 LEU HB2 H 1.590 0.020 1 521 76 76 LEU HB3 H 1.590 0.020 1 522 76 76 LEU HG H 1.590 0.020 1 523 76 76 LEU HD1 H 0.860 0.020 2 524 76 76 LEU HD2 H 0.900 0.020 2 525 76 76 LEU N N 130.055 0.3 1 stop_ save_