Mock Modeling Trial

Mock Modeling Trial

You should first create a homology model of human A2A adenosine receptor in the inactive state based on 2RH1 using Modeller. A2A receptor is involved in regulation of sleep and one of the receptors caffeine binds to. We use Modeller to create homology models. You need to calculate RMSD of your models against the crystal structure of A2A receptor (PDB code: 3EML). Typically we do not create homology models if there is a crystal structure for the protein state of interest but in a mock modeling trial we consider the crystal structure to be the accurate structure of the A2A receptor in our body and see how close our best model is to 3EML. This gives us a measure of modeling success.

3EML shows 4 disulfide bonds (71-159, 74-146, 77-166, 259-262). You should specify these in the Modeller Python file. In the Modeller folder (see below), you see models for the inactive state of AA2A with and without disulfide bonds. You can align these models against the crystal structure 3EML using PyMOL. The python script, findRMSD.py can be run with PyMOL. Download the Modeller folder onto your Desktop from the Google Drive and use the command, 'cd' in PyMOL to move to the downloaded folder on your desktop. Under "File", go to "Run Script" and choose the findRMSD.py file.

After homology modeling with Modeller, you should conduct simulated annealing on Bridges at the Pittsburgh Supercomputing Center to generate many plausible states of the protein to see if you can further improve RMSD against 3EML. To compute the RMSD of the result of simulated annealing to 3eml, you can do RMSD analysis.

We generated simulated annealing (SA) trajectories using GROMACS 2020.4. The starting system is represented by 6.6_equilibration.gro file. This can be loaded onto VMD. If you select just the protein, the coordinates of the protein can be saved as a gro file. This gro file is used to load SA1-protein.xtc and other protein.xtc files. These snapshots can be saved as a pdb file and aligned against 3EML using findRMSD.py file in the Alignment folder within the "With disulfide bonds" simulaiton folder. 

Data from simulated annealing can be found on the Google Drive. (Norimatsu Lab > Molecular Modeling > Mock Modeling > 3EML). The folder called "Without disulfide bonds" is for AA2A without disulfide bonds heated to 400K and 600K (too hot, AA2A denatures). The folder called "With disulfide bond" is for AA2A with disulfide bonds heated to 400K and 500K. The folder called "3EML" is for 3ML crystal structure (as opposed to AA2A homology model). We have simulated annealing runs for 3EML with TM5 and TM6 connected on the intracellular side (created accidentally, might be interesting data) and 3EML with TM5 and TM 6 separated (TM5-6) on the intracellular side. We have SA runs with (TM5-6-DISU) and without (TM5-6) disulfide bonds in 3EML. 

Adenosine A2A - Beta-2 AR Multiple Sequence Alignment:

CLUSTAL O(1.2.4) multiple sequence alignment

ACM1_HUMAN       ---------------------------MN-----T-S--APPAVSPNITVLAPGKGPWQV 25

HRH1_HUMAN       ---------MSLPNSSCLLEDKMCEGNKT-----T-M--ASPQLMPLV------------ 31

ADRB1_HUMAN      MGAGVLVLGASEPGNLSSAAPLP-DGAATAARLLVPASPPASLLPPASESPEPLSQQWTA 59

ADRB2_HUMAN      ---------MGQPGNGSAF-------------LLA----PNGSHAPDHDVTQERDEVWVV 34

DRD2_HUMAN       ----------MDPLNLSWYDDDL---------------ERQNWSRPFNGSDGKADRPHYN 35

AA3R_HUMAN       --------------------------------------------MPNN---STALSLANV 13

AA1R_HUMAN       -----------------------------------------------M---PPSISAFQA 10

AA2AR_HUMAN      -----------------------------------------------------MPIMGSS 7

AA2BR_HUMAN      ----------------------------------------------------MLLETQDA 8

ACM1_HUMAN       AFIGITTGLLSLATVTGNLLVLISFKVNTELKTVNNYFLLSLACADLIIGTFSMNLYTTY 85

HRH1_HUMAN       ----VVLSTICLVTVGLNLLVLYAVRSERKLHTVGNLYIVSLSVADLIVGAVVMPMNILY 87

ADRB1_HUMAN      GM-GLLMALIVLLIVAGNVLVIVAIAKTPRLQTLTNLFIMSLASADLVMGLLVVPFGATI 118

ADRB2_HUMAN      GM-GIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAH 93

DRD2_HUMAN       YY-ATLLTLLIAVIVFGNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYL 94

AA3R_HUMAN       TY-ITMEIFIGLCAIVGNVLVICVVKLNPSLQTTTFYFIVSLALADIAVGVLVMPLAIVV 72

AA1R_HUMAN       AY-IGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCFIVSLAVADVAVGALVIPLAILI 69

AA2AR_HUMAN      VY-ITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITI 66

AA2BR_HUMAN      LY-VALELVIAALSVAGNVLVCAAVGTANTLQTPTNYFLVSLAAADVAVGLFAIPFAITI 67

                          :    :  *:**   .     *:      : **: **: :.   :      

ACM1_HUMAN       LLMGHWALGTLACDLWLALDYVASNASVMNLLLISFDRYFSVTRPLSYRAKR-TPRRAAL 144

HRH1_HUMAN       LLMSKWSLGRPLCLFWLSMDYVASTASIFSVFILCIDRYRSVQQPLRYLKYR-TKTRASA 146

ADRB1_HUMAN      VVWGRWEYGSFFCELWTSVDVLCVTASIETLCVIALDRYLAITSPFRYQSLL-TRARARG 177

ADRB2_HUMAN      ILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLL-TKNKARV 152

DRD2_HUMAN       EVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTV 154

AA3R_HUMAN       SLGITIHF--YSCLFMTCLLLIFTHASIMSLLAIAVDRYLRVKLTVRYKRVT-THRRIWL 129

AA1R_HUMAN       NIGPQTYF--HTCLMVACPVLILTQSSILALLAIAVDRYLRVKIPLRYKMVV-TPRRAAV 126

AA2AR_HUMAN      STGFCAAC--HGCLFIACFVLVLTQSSIFSLLAIAIDRYIAIRIPLRYNGLV-TGTRAKG 123

AA2BR_HUMAN      SLGFCTDF--YGCLFLACFVLVLTQSSIFSLLAVAVDRYLAICVPLRYKSLV-TGTRARG 124

                             * :      :   :*:  :  :..***  :   . *     :  :   

ACM1_HUMAN       MIGLAWLVSFVLWAPA-ILFWQYLVGE-RTV---------LAGQCYI-----QFLSQ--- 185

HRH1_HUMAN       TILGAWFLSFL-WVIP-ILGWNHFMQQTSVR---------REDKCET-----DFYDV--- 187

ADRB1_HUMAN      LVCTVWAISALVSFLPILMHWWRAESDEARRC------YNDPKCC-------DFVTN--R 222

ADRB2_HUMAN      IILMVWIVSGLTSFLPIQMHWYRATHQEAINC------YANETCC-------DFFTN--Q 197

DRD2_HUMAN       MISIVWVLSFTISCPL-LFGLNNADQNEC-----------------------IIANP--- 187

AA3R_HUMAN       ALGLCWLVSFLVGLTP-MFGWNMKLTSEY-H-------------RNVTFLSCQFVSVMRM 174

AA1R_HUMAN       AIAGCWILSFVVGLTP-MFGWNNLSAVER-----AWA---ANGSMGEPVIKCEFEKVISM 177

AA2AR_HUMAN      IIAICWVLSFAIGLTP-MLGWNN--------CGQPKEGKNHSQGCGEGQVACLFEDVVPM 174

AA2BR_HUMAN      VIAVLWVLAFGIGLTP-FLGWNSKDSATN-NCTEPWDGTTNESCC---LVKCLFENVVPM 179

                  :   * ::         :                                  :      

ACM1_HUMAN       PIITFGTAMAAFYLPVTVMCTLYWRIYRETENRARELAALQGS----------------- 228

HRH1_HUMAN       TWFKVMTAIINFYLPTLLMLWFYAKIYKAVRQHCQHRELINRSLPSFSEIKLRPENPKGD 247

ADRB1_HUMAN      AYAI-ASSVVSFYVPLCIMAFVYLRVFREAQKQVKKIDSCERR---FLGG---------- 268

ADRB2_HUMAN      AYAI-ASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGR---FHVQ---------- 243

DRD2_HUMAN       AFVV-YSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVNTKRSSRA-FRAH---------- 235

AA3R_HUMAN       DYMVYFSFLTWIFIPLVVMCAIYLDIFYIIRNKLSLNLSN-------------------- 214

AA1R_HUMAN       EYMVYFNFFVWVLPPLLLMVLIYLEVFYLIRKQLNKKVSA-------------------- 217

AA2AR_HUMAN      NYMVYFNFFACVLVPLLLMLGVYLRIFLAARRQLKQMESQ-------------------- 214

AA2BR_HUMAN      SYMVYFNFFGCVLPPLLIMLVIYIKIFLVACRQLQRTEL--------------------- 218

                       . .  .  *  :   .*  ::    .:                           

ACM1_HUMAN       -ETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQ---------AYSWKE-- 276

HRH1_HUMAN       AKKPGKESPWEVLKRKPKDAGGGSVLKSPSQTPKEMKSPVVFSQEDDREVDKLYCFPLDI 307

ADRB1_HUMAN      P---A------------------------------------------------------- 270

ADRB2_HUMAN      N---L------------------------------------------------------- 245

DRD2_HUMAN       LRAPLKG---------------------NCTHPEDMKLCTVIMKS-------NGSFPVNR 267

AA3R_HUMAN       ------------------------------------------------------------ 214

AA1R_HUMAN       ------------------------------------------------------------ 217

AA2AR_HUMAN      ------------------------------------------------------------ 214

AA2BR_HUMAN      ------------------------------------------------------------ 218

ACM1_HUMAN       --EEEED--------------EGSMES---LTSSEGEE----PG-----SEVVIKMPMVD 308

HRH1_HUMAN       VHMQAAA--------------EGSSRDYVAVNRSHGQLKTDEQGLNTHGASEISEDQMLG 353

ADRB1_HUMAN      ----------------------------RPPSPSPSPVPAPAP----------------- 285

ADRB2_HUMAN      ----------------------------SQV----------------------------- 248

DRD2_HUMAN       RRVEAARRAQELEMEMLSSTSPPERTRYSPIPPSHHQLTLPDPS---------------- 311

AA3R_HUMAN       ------------------------------------------------------------ 214

AA1R_HUMAN       ------------------------------------------------------------ 217

AA2AR_HUMAN      ------------------------------------------------------------ 214

AA2BR_HUMAN      ------------------------------------------------------------ 218

ACM1_HUMAN       PEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKP------------RGKEQLAKRKTFS 356

HRH1_HUMAN       DS-QSFS---RTDSDTTTETAPGKGKLRSGSNTGLDY--IKFTW-KRLRSHSRQYVSGLH 406

ADRB1_HUMAN      --------------PPGPPRPAA------------AAATAPLANGRAG----KRRPSRLV 315

ADRB2_HUMAN      -----------------------------------------EQDGRTGH---GLRRSSKF 264

DRD2_HUMAN       --HHG-----LHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLS 364

AA3R_HUMAN       -------------------------------------------SK----------ETGAF 221

AA1R_HUMAN       ----------------------------------------S--SG----------DPQKY 225

AA2AR_HUMAN      ----------------------------------------PLPGE----------RARST 224

AA2BR_HUMAN      -------------------------------------------MD----------HSRTT 225

ACM1_HUMAN       LVKEKKAARTLSAILLAFILTWTPYNIMVLVSTFCKDC---VPETLWELGYWLCYVNSTI 413

HRH1_HUMAN       MNRERKAAKQLGFIMAAFILCWIPYFIFFMVIAFCKNC---CNEHLHMFTIWLGYINSTL 463

ADRB1_HUMAN      ALREQKALKTLGIIMGVFTLCWLPFFLANVVKAFHR---ELVPDRLFVFFNWLGYANSAF 372

ADRB2_HUMAN      CLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQD---NLIRKEVYILLNWIGYVNSGF 321

DRD2_HUMAN       QQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCD---CNIPPVLYSAFTWLGYVNSAV 421

AA3R_HUMAN       YGREFKTAKSLFLVLFLFALSWLPLSIINCIIYFNG----EVPQLVLYMGILLSHANSMM 277

AA1R_HUMAN       YGKELKIAKSLALILFLFALSWLPLHILNCITLFCPSC--HKPSILTYIAIFLTHGNSAM 283

AA2AR_HUMAN      LQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDC-SHAPLWLMYLAIVLSHTNSVV 283

AA2BR_HUMAN      LQREIHAAKSLAMIVGIFALCWLPVHAVNCVTLFQPAQGKNKPKWAMNMAILLSHANSVV 285

                   :* :  : *  ::  * : * *      .                     : : ** .

ACM1_HUMAN       NPMCYALCNKAFRDTFRLLLLCRWDKRRWRKIP----KRPGSVHRTPSRQC--------- 460

HRH1_HUMAN       NPLIYPLCNENFKKTFKRILHIRS------------------------------------ 487

ADRB1_HUMAN      NPIIYC-RSPDFRKAFQRLLCCARRAARRR--HA--------THGDRPRASGCLARPGPP 421

ADRB2_HUMAN      NPLIYC-RSPDFRIAFQELLCLRRSSLKAY--GNGY-----SSNGNTGEQSGYHVEQ--- 370

DRD2_HUMAN       NPIIYTTFNIEFRKAFLKILHC-------------------------------------- 443

AA3R_HUMAN       NPIVYAYKIKKFKETYLLILKACVVCHPSDSLDTSIEKNSE------------------- 318

AA1R_HUMAN       NPIVYAFRIQKFRVTFLKIWNDHFRCQPAPPIDEDLPEE--------------------- 322

AA2AR_HUMAN      NPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPP 343

AA2BR_HUMAN      NPIVYAYRNRDFRYTFHKIISRYLLCQADVKSGNGQ----------AGVQPALGVGL--- 332

                 **: *      *: ::  :                                         

ACM1_HUMAN       ------------------------------------------------------------ 460

HRH1_HUMAN       ------------------------------------------------------------ 487

ADRB1_HUMAN      --------PSPGAASDD---------------------------DDDDVVGATPPARLLE 446

ADRB2_HUMAN      ----------------E---------------------------KENKLLCEDLP--GTE 385

DRD2_HUMAN       ------------------------------------------------------------ 443

AA3R_HUMAN       ------------------------------------------------------------ 318

AA1R_HUMAN       -------------RPDD------------------------------------------- 326

AA2AR_HUMAN      GVWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPEPP--GLD 401

AA2BR_HUMAN      ------------------------------------------------------------ 332

ACM1_HUMAN       ------------------------------- 460

HRH1_HUMAN       ------------------------------- 487

ADRB1_HUMAN      PWAGCNGGAAADSDSSLDEPCRPGFASESKV 477

ADRB2_HUMAN      DFVGHQGTVPSDNIDSQGRNCSTND---SLL 413

DRD2_HUMAN       ------------------------------- 443

AA3R_HUMAN       ------------------------------- 318

AA1R_HUMAN       ------------------------------- 326

AA2AR_HUMAN      DPLAQDGAGVS-------------------- 412

AA2BR_HUMAN      ------------------------------- 332