data_27202 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; Extrinsic methylthiocysteine labels of cytosolic domain of NADPH cytochrome P450 oxidoreductase ; _BMRB_accession_number 27202 _BMRB_flat_file_name bmr27202.str _Entry_type original _Submission_date 2017-08-06 _Accession_date 2017-08-06 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details ; Chemical shift assignment of extrinsic methylthiocysteine labels in NADPH cytochrome P450 oxidoreductase. Into original cysteine-less construct mutations Q157C and Q517C were introduced via mutagenesis. Later on those cysteines were labeled with methyl-methanethiosulfonate to result methylthiocysteine groups. ; loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Galiakhmetov Azamat R. . 2 Kovrigin Evgueni L. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 2 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 5 "13C chemical shifts" 6 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2018-03-28 update BMRB 'update entry citation' 2017-12-11 original author 'original release' stop_ _Original_release_date 2017-08-10 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title ; Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: 13C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 29168021 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Galiakhmetov Azamat R. . 2 Kovrigina Elizaveta A. . 3 Xia Chuanwu . . 4 Kim Jung-Ja . . 5 Kovrigin Evgueni L. . stop_ _Journal_abbreviation 'J. Biomol. NMR' _Journal_volume 70 _Journal_issue 1 _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first 21 _Page_last 31 _Year 2018 _Details . save_ ################################## # Molecular system description # ################################## save_assembly _Saveframe_category molecular_system _Mol_system_name POR _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label POR $oxidoreductase stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state ? _System_paramagnetic yes _System_thiol_state . _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_oxidoreductase _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common oxidoreductase _Molecular_mass . _Mol_thiol_state 'all other bound' _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 622 _Mol_residue_sequence ; IQTTAPPVKESSFVEKMKKT GRNIIVFYGSQTGTAEEFAN RLSKDAHRYGMRGMSADPEE YDLADLSSLPEIDKSLVVFC MATYGEGDPTDNAQDFYDWL QETDVDLTGVKFAVFGLGNK TYEHFNAMGKYVDQRLEQLG AQRIFELGLGDDDGNLEEDF ITWREQFWPAVCEFFGVEAT GEESSIRQYELVVHEDMDVA KVYTGEMGRLKSYENQKPPF DAKNPFLAAVTANRKLNQGT ERHLMHLELDISDSKIRYES GDHVAVYPANDSALVNQIGE ILGADLDVIMSLNNLDEESN KKHPFPCPTTYRTALTYYLD ITNPPRTNVLYELAQYASEP SEQEHLHKMASSSGEGKELY LSWVVEARRHILAILQDYPS LRPPIDHLCELLPRLQARYY SIASSSKVHPNSVHICAVAV EYEAKSGRVNKGVATSWLRA KEPAGENGGRALVPMFVRKS QFRLPFKSTTPVIMVGPGTG IAPFMGFIQERAWLREQGKE VGETLLYYGCRRSDEDYLYR EELARFHKDGALTQLNVAFS REQAHKVYVQHLLKRDREHL WKLIHEGGAHIYVCGDARNM AKDVQNTFYDIVAEFGPMEH TQAVDYVKKLMTKGRYSLDV WS ; loop_ _Residue_seq_code _Residue_label 1 ILE 2 GLN 3 THR 4 THR 5 ALA 6 PRO 7 PRO 8 VAL 9 LYS 10 GLU 11 SER 12 SER 13 PHE 14 VAL 15 GLU 16 LYS 17 MET 18 LYS 19 LYS 20 THR 21 GLY 22 ARG 23 ASN 24 ILE 25 ILE 26 VAL 27 PHE 28 TYR 29 GLY 30 SER 31 GLN 32 THR 33 GLY 34 THR 35 ALA 36 GLU 37 GLU 38 PHE 39 ALA 40 ASN 41 ARG 42 LEU 43 SER 44 LYS 45 ASP 46 ALA 47 HIS 48 ARG 49 TYR 50 GLY 51 MET 52 ARG 53 GLY 54 MET 55 SER 56 ALA 57 ASP 58 PRO 59 GLU 60 GLU 61 TYR 62 ASP 63 LEU 64 ALA 65 ASP 66 LEU 67 SER 68 SER 69 LEU 70 PRO 71 GLU 72 ILE 73 ASP 74 LYS 75 SER 76 LEU 77 VAL 78 VAL 79 PHE 80 CYS 81 MET 82 ALA 83 THR 84 TYR 85 GLY 86 GLU 87 GLY 88 ASP 89 PRO 90 THR 91 ASP 92 ASN 93 ALA 94 GLN 95 ASP 96 PHE 97 TYR 98 ASP 99 TRP 100 LEU 101 GLN 102 GLU 103 THR 104 ASP 105 VAL 106 ASP 107 LEU 108 THR 109 GLY 110 VAL 111 LYS 112 PHE 113 ALA 114 VAL 115 PHE 116 GLY 117 LEU 118 GLY 119 ASN 120 LYS 121 THR 122 TYR 123 GLU 124 HIS 125 PHE 126 ASN 127 ALA 128 MET 129 GLY 130 LYS 131 TYR 132 VAL 133 ASP 134 GLN 135 ARG 136 LEU 137 GLU 138 GLN 139 LEU 140 GLY 141 ALA 142 GLN 143 ARG 144 ILE 145 PHE 146 GLU 147 LEU 148 GLY 149 LEU 150 GLY 151 ASP 152 ASP 153 ASP 154 GLY 155 ASN 156 LEU 157 GLU 158 GLU 159 ASP 160 PHE 161 ILE 162 THR 163 TRP 164 ARG 165 GLU 166 GLN 167 PHE 168 TRP 169 PRO 170 ALA 171 VAL 172 CYS 173 GLU 174 PHE 175 PHE 176 GLY 177 VAL 178 GLU 179 ALA 180 THR 181 GLY 182 GLU 183 GLU 184 SER 185 SER 186 ILE 187 ARG 188 GLN 189 TYR 190 GLU 191 LEU 192 VAL 193 VAL 194 HIS 195 GLU 196 ASP 197 MET 198 ASP 199 VAL 200 ALA 201 LYS 202 VAL 203 TYR 204 THR 205 GLY 206 GLU 207 MET 208 GLY 209 ARG 210 LEU 211 LYS 212 SER 213 TYR 214 GLU 215 ASN 216 GLN 217 LYS 218 PRO 219 PRO 220 PHE 221 ASP 222 ALA 223 LYS 224 ASN 225 PRO 226 PHE 227 LEU 228 ALA 229 ALA 230 VAL 231 THR 232 ALA 233 ASN 234 ARG 235 LYS 236 LEU 237 ASN 238 GLN 239 GLY 240 THR 241 GLU 242 ARG 243 HIS 244 LEU 245 MET 246 HIS 247 LEU 248 GLU 249 LEU 250 ASP 251 ILE 252 SER 253 ASP 254 SER 255 LYS 256 ILE 257 ARG 258 TYR 259 GLU 260 SER 261 GLY 262 ASP 263 HIS 264 VAL 265 ALA 266 VAL 267 TYR 268 PRO 269 ALA 270 ASN 271 ASP 272 SER 273 ALA 274 LEU 275 VAL 276 ASN 277 GLN 278 ILE 279 GLY 280 GLU 281 ILE 282 LEU 283 GLY 284 ALA 285 ASP 286 LEU 287 ASP 288 VAL 289 ILE 290 MET 291 SER 292 LEU 293 ASN 294 ASN 295 LEU 296 ASP 297 GLU 298 GLU 299 SER 300 ASN 301 LYS 302 LYS 303 HIS 304 PRO 305 PHE 306 PRO 307 CYS 308 PRO 309 THR 310 THR 311 TYR 312 ARG 313 THR 314 ALA 315 LEU 316 THR 317 TYR 318 TYR 319 LEU 320 ASP 321 ILE 322 THR 323 ASN 324 PRO 325 PRO 326 ARG 327 THR 328 ASN 329 VAL 330 LEU 331 TYR 332 GLU 333 LEU 334 ALA 335 GLN 336 TYR 337 ALA 338 SER 339 GLU 340 PRO 341 SER 342 GLU 343 GLN 344 GLU 345 HIS 346 LEU 347 HIS 348 LYS 349 MET 350 ALA 351 SER 352 SER 353 SER 354 GLY 355 GLU 356 GLY 357 LYS 358 GLU 359 LEU 360 TYR 361 LEU 362 SER 363 TRP 364 VAL 365 VAL 366 GLU 367 ALA 368 ARG 369 ARG 370 HIS 371 ILE 372 LEU 373 ALA 374 ILE 375 LEU 376 GLN 377 ASP 378 TYR 379 PRO 380 SER 381 LEU 382 ARG 383 PRO 384 PRO 385 ILE 386 ASP 387 HIS 388 LEU 389 CYS 390 GLU 391 LEU 392 LEU 393 PRO 394 ARG 395 LEU 396 GLN 397 ALA 398 ARG 399 TYR 400 TYR 401 SER 402 ILE 403 ALA 404 SER 405 SER 406 SER 407 LYS 408 VAL 409 HIS 410 PRO 411 ASN 412 SER 413 VAL 414 HIS 415 ILE 416 CYS 417 ALA 418 VAL 419 ALA 420 VAL 421 GLU 422 TYR 423 GLU 424 ALA 425 LYS 426 SER 427 GLY 428 ARG 429 VAL 430 ASN 431 LYS 432 GLY 433 VAL 434 ALA 435 THR 436 SER 437 TRP 438 LEU 439 ARG 440 ALA 441 LYS 442 GLU 443 PRO 444 ALA 445 GLY 446 GLU 447 ASN 448 GLY 449 GLY 450 ARG 451 ALA 452 LEU 453 VAL 454 PRO 455 MET 456 PHE 457 VAL 458 ARG 459 LYS 460 SER 461 GLN 462 PHE 463 ARG 464 LEU 465 PRO 466 PHE 467 LYS 468 SER 469 THR 470 THR 471 PRO 472 VAL 473 ILE 474 MET 475 VAL 476 GLY 477 PRO 478 GLY 479 THR 480 GLY 481 ILE 482 ALA 483 PRO 484 PHE 485 MET 486 GLY 487 PHE 488 ILE 489 GLN 490 GLU 491 ARG 492 ALA 493 TRP 494 LEU 495 ARG 496 GLU 497 GLN 498 GLY 499 LYS 500 GLU 501 VAL 502 GLY 503 GLU 504 THR 505 LEU 506 LEU 507 TYR 508 TYR 509 GLY 510 CYS 511 ARG 512 ARG 513 SER 514 ASP 515 GLU 516 ASP 517 TYR 518 LEU 519 TYR 520 ARG 521 GLU 522 GLU 523 LEU 524 ALA 525 ARG 526 PHE 527 HIS 528 LYS 529 ASP 530 GLY 531 ALA 532 LEU 533 THR 534 GLN 535 LEU 536 ASN 537 VAL 538 ALA 539 PHE 540 SER 541 ARG 542 GLU 543 GLN 544 ALA 545 HIS 546 LYS 547 VAL 548 TYR 549 VAL 550 GLN 551 HIS 552 LEU 553 LEU 554 LYS 555 ARG 556 ASP 557 ARG 558 GLU 559 HIS 560 LEU 561 TRP 562 LYS 563 LEU 564 ILE 565 HIS 566 GLU 567 GLY 568 GLY 569 ALA 570 HIS 571 ILE 572 TYR 573 VAL 574 CYS 575 GLY 576 ASP 577 ALA 578 ARG 579 ASN 580 MET 581 ALA 582 LYS 583 ASP 584 VAL 585 GLN 586 ASN 587 THR 588 PHE 589 TYR 590 ASP 591 ILE 592 VAL 593 ALA 594 GLU 595 PHE 596 GLY 597 PRO 598 MET 599 GLU 600 HIS 601 THR 602 GLN 603 ALA 604 VAL 605 ASP 606 TYR 607 VAL 608 LYS 609 LYS 610 LEU 611 MET 612 THR 613 LYS 614 GLY 615 ARG 616 TYR 617 SER 618 LEU 619 ASP 620 VAL 621 TRP 622 SER stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date . save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $oxidoreductase Rat 10116 Eukaryota Metazoa Rattus norvegicus 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 $oxidoreductase 'recombinant technology' . Escherichia coli . pOR263 stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details 'NADPH cytochrome P450 oxidoreductase' loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $oxidoreductase . mM . 'sodium phosphate' 1 mM 'natural abundance' 'potassium phosphate' 10 mM 'natural abundance' 'sodium chloride' 137 mM 'natural abundance' 'potassium chloride' 2.7 mM 'natural abundance' stop_ save_ ############################ # Computer software used # ############################ 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 600 _Details 'Ignore INOVA; this was VNMR-S' save_ save_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model Avance _Field_strength 600 _Details . save_ ############################# # NMR applied experiments # ############################# save_2D_1H-13C_HMQC_1 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-13C HMQC' _Sample_label $sample_1 save_ ####################### # Sample conditions # ####################### save_methylthiocysteine _Saveframe_category sample_conditions _Details '13C methyl group attached to surface exposed cysteine via disulfide bond' loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 100 . mM pH 7.4 . pH pressure 1 . atm temperature 293 . 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 na C 13 'methyl carbon' ppm 0 na indirect . . . 0.25144953 water H 1 protons ppm 4.8 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 'There is a peak for 308 PRO H at 2.490.' loop_ _Experiment_label '2D 1H-13C HMQC' stop_ loop_ _Sample_label $sample_1 stop_ _Sample_conditions_label $methylthiocysteine _Chem_shift_reference_set_label $chemical_shift_reference_1 _Mol_system_component_name POR _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 1 127 127 ALA H H 2.409 . . 2 127 127 ALA C C 25.276 . . 3 157 157 GLU H H 2.138 . . 4 157 157 GLU C C 23.687 . . 5 308 308 PRO C C 24.246 . . 6 517 517 TYR H H 2.537 . . 7 517 517 TYR C C 24.809 . . stop_ save_ save_assigned_chem_shift_list_2 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Experiment_label '2D 1H-13C HMQC' stop_ loop_ _Sample_label $sample_1 stop_ _Sample_conditions_label $methylthiocysteine _Chem_shift_reference_set_label $chemical_shift_reference_1 _Mol_system_component_name POR _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 1 157 157 GLU H H 2.156 . . 2 157 157 GLU C C 23.711 . . 3 517 517 TYR H H 2.532 . . 4 517 517 TYR C C 25.023 . . stop_ save_