Homology based structure predictions

Homologous

Because we found no homologous structures in Task 2, we extended our list by using HHSearch.

HHSearch found just sequences with an indentity below 40% therefore we will use the 12 proteins shown below for creating a multiple alignment for homologous modeling. We choose sequences to cover the whole protein and we pay specific attention on the transmembrane region.

PDB-ID	Identity	Description
1s79	37%	human La protein
3p73	28%	classical MHC class I molecule
1kcg	22%	NKG2D
1jfm	14%	MURINE NK CELL LIGAND RAE-1 BETA
1bii	22%	H-2DD MHC CLASS I
2p24	21%	alphabeta TCR
1cd1	21%	MHC-like fold with a large hydrophobic binding groove
2wy3	29%	HCMV UL16-MICB complex
1lqv	14%	Endothelial protein C receptor
3jts	25%	Mamu A*2
1ow0	22%	human FcaRI
1hxm	18%	Human Vgamma9/Vdelta2 T Cell Receptor

With these Sequences including the HFE-Gen(Q30201), we did a multiple sequence alignment with t-coffee(EXPRESSO). This multiple sequence alignment is later used as a raw alignment in the Alignment Mode of SwissModel and Modeller. Later on, we will try to fit better models by editing the alignment by keeping functional regions together.

  DSSP                                   --EEEEEEEEEEB-SS-SSB--EEEEEETTEEEEEEESSS--EEE--STTS-SSTTTTHHHHHHHHHHHHHHHHH
Q30201          MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHES--RRVE-PRTPWVSSRISSQMWLQLSQSLKGWDHM
1S79_A          --------------------------------------------------------------------GRW-IL-KNDVKNRSVYIKGFPTDATLDDIKE
3P73_A          -----------------------EFGSHSLRYFLTGMTDPGPGMPRFVIVGYVDDKIFGTYNSKS--RTAQ-PIVEML-PQEDQEHWDTQTQKAQGGERD
1KCG_C          -------------------------DAHSLWYNFTIIHLPRHGQQWCEVQSQVDQKNFLSYDCGS--DKVLSMGHL-EEQLYATDAWGKQLEMLREVGQR
1JFM_A          -------------------------DAHSLRCNLTIKDPTPADPLWYEAKCFVGEILILHLSNIN--KTMT-SG-DPGETANATEVKKCLTQPLKNLCQK
1BII_A          -MGAMAPRTLLLLLAAALGPTQTRAGSHSLRYFVTAVSRPGFGEPRYMEVGYVDNTEFVRFDSDAENPRYE-PRARWIE-QEGPEYWERETRRAKGNEQS
2P24_A          ----------------------------------------------------------------------------M----AIMAPRTLVLLLSGALALT
1CD1_A          -----------------------QQKNYTFRCLQMSSFANR-SWSRTDSVVWLGDLQTHRWSNDS--ATIS-FTKPWSQGKLSNQQWEKLQHMFQVYRVS
2WY3_A          ------------------------MEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQK--RRAK-PQGQWAEDVLGAETWDTETEDLTENGQD
1LQV_A          -------------------SQDASDGLQRLHMLQISYFR-DPYHVWYQGNASLGGHLTHVLEGPDTNTTII-QLQPL----QEPESWARTQSGLQSYLLQ
3JTS_A          -------------------------GSHSMRYFYTSMSRPGRWEPRFIAVGYVDDTQFVRFDSDAASQRME-PRAPWVE-QEGPEYWDRETRNMKAETQN
1OW0_A          ----------------------------------------------------------------------------------------------------
1HXM_A          -------------------------------------------------------------------------------------AIELVPEHQTVPVSI
                                                                 
  DSSP          HHHHHHHHTTT-SSS--E--------EEEEEE-EEE-TTS-E-EEE-E------------EEEETTEE----------------EEEEEGGGTEEEES--
Q30201          FTVDFWTIMENHN-HSKE--------SHTLQV-ILGCEMQED-NST-E------------GYWKYGYD----------------GQDHLEFCPDTLDW--
1S79_A          WLEDKGQV-LNIQMRRTL--------HKAFKG-SIFVVFDSI-ESA-KKFVETPGQKYKETDLLILFKDDYFAKKNEERKQNKVE---------------
3P73_A          FDWNLNRLPERYN-KSKG--------SHTMQM-MFGCDILED-GSI-R------------GYDQYAFD----------------GRDFLAFDMDTMTF--
1KCG_C          LRLELADT---------ELEDFTPSGPLTLQV-RMSCECEAD-GYI-R------------GSWQFSFD----------------GRKFLLFDSNNRKW--
1JFM_A          LRNKVSNT-KVDTHKTNG--------YPHLQV-TMIYPQSQG-RTP-S------------ATWEFNIS----------------DSYFFTFYTENMSW--
1BII_A          FRVDLRTALRYYNQSAGG--------SHTLQW-MAGCDVESD-GRLLR------------GYWQFAYD----------------GCDYIALNEDLKTW--
2P24_A          QTWAGSHSRGEDD--IEA--------DHVGSYGIVVYQSP----GD-I------------GQYTFEFD----------------GDELFYVDLDKKET--
1CD1_A          FTRDIQELVKMMSPKEDY--------PIEIQL-SAGCEMYPG-NAS-E------------SFLHVAFQ----------------GKYVVRFWG--TSWQT
2WY3_A          LRRTLTHI----KDQKGG--------LHSLQE-IRVCEIHED-SST-R------------GSRHFYYN----------------GELFLSQNLETQES--
1LQV_A          FHGLVRLVHQERT--LAF--------PLTIRC-FLGCELPPEGSRA-H------------VFFEVAVN----------------GSSFVSFRPERALW--
3JTS_A          APVNLRNLRGYYNQSEAG--------SHTIQR-MYGCDLGPD-GRLLR------------GYHQSAYD----------------GKDYIALNEDLRSW--
1OW0_A          -----ACHPRLSLHRPAL--------EDLLLG-SEANLTCTL-TGLRD------------ASGVTFTW----------------TPSSGKSAV--QGPPE
1HXM_A          GVPATLRCSMKGEAIGNY--------YINWYR-KTQGNTMTF-IYRE-------------KDIYGPGF----------------KDNFQGDIDIAKNL--
                                                                 
  DSSP          SGG-G----HHH-HHHHHSSTHHH--HHHHHHHHTHHHHHHHHHHHHHTTTSS--B--EEEEEEEE-SS-----E-EEEEEEEEEBSS--EEEEEETTEE
Q30201          RAA-E----PRA-WPTKLEWERHK--IRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVT----S-SVTTLRCRALNYYPQNITMKWLKD
1S79_A          ----------------------------------------------------------------------------------------------------
3P73_A          TAA-D----PVA-EITKRRWETEG--TYAERWKHELGTVCVQNLRRYLEHGKAALKRRVQPEVRVWGKEA----D-GILTLSCHAHGFYPRPITISWMKD
1KCG_C          TVV-H----AGA-RRMKEKWEKDS--GLTTFFKMVSMRDCKSWLRDFLMHRKKRLE--------------------------------------------
1JFM_A          RSA-N----DES-GVIMNKWKDDG--EFVKQLKFLI-HECSQKMDEFLKQSKEK----------------------------------------------
1BII_A          TAA-D----MAA-QITRRKWEQA---GAAERDRAYLEGECVEWLRRYLKNGNATLLRTDPPKAHVTHHRR----PEGDVTLRCWALGFYPADITLTWQLN
2P24_A          IWM-------------LPEFAQLR--SFDPQGGLQNIATGKHNLGVLTKRSNSTPATNEAPQATVFPKSP--VLLGQPNTLICFVDNIFPPVINITWLRN
1CD1_A          VPGAP----SWL-DLPIKVLNADQ--GTSATVQMLLNDTCPLFVRGLLEAGKSDLEKQEKPVAWLSSVP---SSAHGHRQLVCHVSGFYPKPVWVMWMRG
2WY3_A          TVP-QSSRAQTLAMNVTNFW-KEDAMKTKTHYRAMQ-ADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVS----EGNITVTCRASSFYPRNITLTWRQDG
1LQV_A          QAD-TQVTSGVV-TFTLQQLNAYN--RTRYELREFLEDTCVQYVQKHISAENTKGSQTSRSYTS------------------------------------
3JTS_A          TAA-D----MAA-QNTQRKWEAA---GEAEQHRTYLEGECLEWLRRYLENGKETLQRADPPKTHVTHHPV----SDQEATLRCWALGFYPAEITLTWQRD
1OW0_A          R--DL----CGC-YSVSSVLPGCA--EPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQG
1HXM_A          AVL-K----ILA-PSERDEGSYYC--ACDTLGMGGEYTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNG---------TNVACLVKEFYPKDIRINLVSS
                                                                  
  DSSP          --GGGS---EEEE-TTS-E----EEEEEEEE-TTGGGGEE---EEEE-TTSSS-EEE-E-
Q30201          K-QPMDAKEFEPKDVLPNG----DGTYQGWITLAVPPGEE---QRYTCQVEHPGLDQ-PLIVIWEPSPSGTLVIGVISGIAVFVVILFIGILFIILRKRQ
1S79_A          ----------------------------------------------------------------------------------------------------
3P73_A          --GMVRDQETRWGGIVPNS----DGTYHASAAIDVLPEDG---DKYWCRVEHASLPQ-PGLFSWEPQ---------------------------------
1KCG_C          ----------------------------------------------------------------------------------------------------
1JFM_A          ----------------------------------------------------------------------------------------------------
1BII_A          --GEELTQEMELVETRPAG----DGTFQKWASVVVPLGKE---QKYTCHVEHEGLPE-PLTLRWGKEEPPSSTKTNTVIIAVPVVLGAVVILGAVMAFVM
2P24_A          --SKSVADGVYETSFFVNR----DYSFHKLSYLTFIPSDD---DIYDCKVEHWGLEE-PVLKHWEPEIPAPMSELTETSGSRLEVLFQ------------
1CD1_A          --DQ-EQQGTHRGDFLPNA----DETWYLQATLDVEAGEE---AGLACRVKHSSLGG-QDIILYWDARQAPVGLIVFIVLIMLVVVGAVVYYIWRRRSAY
2WY3_A          --VSLSHNTQQWGDVLPDG----NGTYQTWVATRIRQGEE---QRFTCYMEHSGNHG-THPVPSGKVLVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKK
1LQV_A          ----------------------------------------------------------------------------------------------------
3JTS_A          --GEDQTQDTELVETRPAG----DGTFQKWAAVVVPSGKE---QRYTCHVQHEGLRE-PLTLRWEP----------------------------------
1OW0_A          SQEL-PREKYLTW-ASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGK------------------------------
1HXM_A          -----KKITEFDPAIVISP----SGKYNAVKLGKYE--DS---NSVTCSVQHDNK---TVHSTDFEVKTDSTDHVKPKETENTKQPSKS-----------
                                                                  
  DSSP
Q30201          GSRGAMGHYVLAERE----------------
1S79_A          -------------------------------
3P73_A          -------------------------------
1KCG_C          -------------------------------
1JFM_A          -------------------------------
1BII_A          KRRRNTGGKGGDYALAPGSQSSDMSLPDCKV
2P24_A          -------------------------------
1CD1_A          QDIR---------------------------
2WY3_A          KTSAAEGP-----------------------
1LQV_A          -------------------------------
3JTS_A          -------------------------------
1OW0_A          -------------------------------
1HXM_A          -------------------------------

Based on the secondary structure for the HFE-Gen assigned by DSSP from the PDB structure (1a6z) the multible sequence alignmet conserves most parts of the secondary structure.

I-Tasser

perfomance of I-TASSER at CASP
Source: http://zhanglab.ccmb.med.umich.edu/I-TASSER/about.html

Workflow of the I-Tasser server
Source: http://zhanglab.ccmb.med.umich.edu/I-TASSER/about.html

I-Tasser is a webservice for protein structure prediction provided and published by Ambrish Roy, Alper Kucukural and Yang Zhang at http://zhanglab.ccmb.med.umich.edu/I-TASSER/ for the CASP competition with outstanding achievement.

The I-Tasser protocol consists of serveral steps which are:

• threading the seqeunce into different structure to create an initial template.
• break the template apart into fragments which matched the structure (leave out the parts of the structrue to which no sequence is assigned).
• Structure assembly and clustering
• use the cluster centroid for structure reassembly
• search the structure with the lowest energie and do REMO H-bond optimization to get the final model.

Further on, I-Tasser also predict GO-Terms and binding sites. Therfore it use the final model to search for global and local matches in the PDB to predict these terms.

For us, a problem is that I-Tasser only provieds complete models, but the PDB structure of our protein isn't complete. Threrefore we used DSSP to compare the secondary structure of all models with the one form UniProt and PDB. TODO!!

Predicted Secondary Structure by I-Tasser

Sequence:   MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDF
Predicted:  CCCCHHHHHHHHHHHHHHHHHHHHCCCCCCCSSSSSCCCCCCCCCCSSSSSSSCCCSSSSCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHH
Conf-Score: 985028899999999899875122045421036641367999985269985643743686068998778788540145583478888887676654315558

Sequence:   WTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQ
Predicted:  HHHHHHHCCCCCCSSSSSSSCCCCCCCCCCCCCCCCCCCCCCSSSSCCCHHHCHHHHHHHHHHHHHHHHCCCHHHHHHHHHCCCCHHHHHHHHHCCHHHHHC
Conf-Score: 888755315777644463525565898763541000558873365263022202455666677878887004598888767064299999999747666642

Sequence:   QVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLV
Predicted:  CCCCCCCCCCCCCCCHHHHCHHHHCCCCCCSSSSSSSCCCCCCCCCCSSSSCCCCCCCCCCCSSSSSCCCCCCCCSSSSCCCCCCCCCSSSSCCCCCCCCCC
Conf-Score: 599877567699854442101541541332479864358754456553541024888652112699807986310267512589998726840688766531

Sequence:   IGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAERE
Prediction: CCCCCCHHHHHHHCCHHHHHHHHHCCCCCCCCCCCCCHCCCC
Conf-Score: 010211112222100246665443013678898651020169

Secondary structure elements are shown as H for Alpha helix,S for Beta sheet & C for Coil

Predicted Solvent Accessibility by I-Tasser

Sequence:   MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDF
Prediction: 723312000000000101112222011200120120023333331200000102322003123724434241311436413610352044144313323230

Sequence:   WTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQ
Prediction: 220132133351310001010021136231211333023032003016303403102321432433044143404422010333005103400630351154

Sequence:   QVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLV
Prediction: 342353313321443300000100101014010203346564435434135233334221320000000347533120214264144202020214542200

Sequence:   IGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAERE
Prediction: 000001100000011100000001334446443132333438

Values range from 0 (buried residue) to 9 (highly exposed residue)

I-Tasser predicted five Models with a C-Score from -0.557 to -3.298. They are ranked from one to five as seen below. As cutoff for the C-Score, we use -1.5 as recommended by the Zhang group<ref>Ambrish Roy, Alper Kucukural, Yang Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, vol 5, 725-738 (2010). | Zhang et al.</ref> that is proposed to give a false-positive and false-negative rate of about 0.1. That means more than 90% of the quality predictions are correct. Therfore we just use Model1 for the comparison with the other methods.

Model 1 with a C-Score of -0.557

Model 2 with a C-Score of -2.539

Model 3 with a C-Score of -2.266

Model 4 with a C-Score of -2.772

Model 5 with a C-Score of -3.298

Seq:  MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMEN
Pred: CCCCHHHHHHHHHHHHHHHHHHHHCCCCCCCEEEEECCCCCCCCCCEEEEEEECCCEEEECCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHH
UniP: ---------------------------EEEEEEEEEEE----EEE--EEEEEE--EEEEEEEEEE--EEE--------TTTHHHHHHHHHHHHHHHHHHHHHHHHHHT

Seq:  HNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHV
Pred: HCCCCCCEEEEEEECCCCCCCCCCCCCCCCCCCCCCEEEECCCHHHCHHHHHHHHHHHHHHHHCCCHHHHHHHHHCCCCHHHHHHHHHCCHHHHHCCCCCCCCCCCCC
UniP: TT-EEE--EEEEEEEEEE-----EEEEEEEEE--EEEEEEEHHH-EEEEEE---HHHHHHHH---HHHHHHHHHHH-HHHHHHHHHHHHHTTT-------EEEEEEEE

Seq:  TSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLVIGVISGIAVFVVILFIGI
Pred: CCCHHHHCHHHHCCCCCCEEEEEEECCCCCCCCCCEEEECCCCCCCCCCCEEEEECCCCCCCCEEEECCCCCCCCCEEEECCCCCCCCCCCCCCCCHHHHHHHCCHHH
UniP: ----EEEEEEEEEEEEE--EEEEEE------HHH----EEEE-----EEEEEEEEE---HHHHEEEEEE---EEE-EEEE----------------------------

Seq:  LFIILRKRQGSRGAMGHYVLAERE
Pred: HHHHHHCCCCCCCCCCCCCHCCCC
UniP: ------------------------

For a better overview we replaced the I-Tasser S for Sheet by an E like in the UniProt secondary structure.

Model1 has a TM-Score of about 0.64 and a RMSD of 7.7Å. For the prediction, I-Tasser used 1a6zA, 1s7qA, 1i4fA, 1de4A, 2vabA and 2bckA as templates. The templates have an identity of about 40% except for the self hit 1a6z. Because of the self hit, we run I-Tasser a second time with the constrain to exclude all templates with a sequence identity > 80%.

I-Tasser using templates with a sequence identity below 80% to avoid self hits.

SwissModel

SwissProt is a server based tool provided by the SIB. It combines tools like PSI-PRED and DISOPRED for secondary structure and disordered region prediction.

The model created by SwissModel is based on a self hit, but we had no chance to exclude the protein itself from the prediction. Therefore we also run SwissModel in Alignment-Mode.(TODO)

Automated Mode

predicted model

Model information: Modelled residue range: 26 to 297
Based on template: 1a6zC (2.60 Å)
Sequence Identity [%]: 100
Evalue: 7.66e-163

Quality information: QMEAN Z-Score: -1.035

Estimated absolute model quality

Estimated density of model quality

Z-Score by category

predicted error

Even though the model is based on a self hit, the Z-Score is about -1, which means that the model is one standard deviation from the mean. The model is not quite unlikely but also not the most probable one.

References

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