+
データを開く
-
基本情報
登録情報 | データベース: EMDB / ID: EMD-6902 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
タイトル | Yeast TRiC/CCT with PA tag insertion in CCT6 subunits | |||||||||
![]() | Yeast TRiC/CCT with PA tag in CCT6 subunits | |||||||||
![]() |
| |||||||||
機能・相同性 | ![]() Association of TriC/CCT with target proteins during biosynthesis / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / chaperonin-containing T-complex / : / Neutrophil degranulation / ATP-dependent protein folding chaperone / unfolded protein binding / protein folding / ATP hydrolysis activity / ATP binding ...Association of TriC/CCT with target proteins during biosynthesis / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / chaperonin-containing T-complex / : / Neutrophil degranulation / ATP-dependent protein folding chaperone / unfolded protein binding / protein folding / ATP hydrolysis activity / ATP binding / plasma membrane / cytoplasm 類似検索 - 分子機能 | |||||||||
生物種 | ![]() ![]() ![]() ![]() | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 11.4 Å | |||||||||
![]() | Wang H / Takagi J / Cong Y | |||||||||
![]() | ![]() タイトル: Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis. 著者: Huping Wang / Wenyu Han / Junichi Takagi / Yao Cong / ![]() ![]() 要旨: Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural ...Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes. | |||||||||
履歴 |
|
-
構造の表示
ムービー |
![]() |
---|---|
構造ビューア | EMマップ: ![]() ![]() ![]() |
添付画像 |
-
ダウンロードとリンク
-EMDBアーカイブ
マップデータ | ![]() | 59.9 MB | ![]() | |
---|---|---|---|---|
ヘッダ (付随情報) | ![]() ![]() | 16.9 KB 16.9 KB | 表示 表示 | ![]() |
画像 | ![]() | 115.3 KB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
-
リンク
EMDBのページ | ![]() ![]() |
---|---|
「今月の分子」の関連する項目 |
-
マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
注釈 | Yeast TRiC/CCT with PA tag in CCT6 subunits | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.3 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
|
-添付データ
-
試料の構成要素
-全体 : Yeast TRiC/CCT with PA tag in CCT6 subunits
全体 | 名称: Yeast TRiC/CCT with PA tag in CCT6 subunits |
---|---|
要素 |
|
-超分子 #1: Yeast TRiC/CCT with PA tag in CCT6 subunits
超分子 | 名称: Yeast TRiC/CCT with PA tag in CCT6 subunits / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all / 詳細: TRiC incubation with PA tag antibody NZ-1 |
---|---|
由来(天然) | 生物種: ![]() ![]() |
組換発現 | 生物種: ![]() ![]() |
分子量 | 理論値: 1 MDa |
-分子 #1: T-complex protein 1 subunit alpha
分子 | 名称: T-complex protein 1 subunit alpha / タイプ: protein_or_peptide / ID: 1 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MSQLFNNSRS DTLFLGGEK I SGDDIRNQ NV LATMAVA NVV KSSLGP VGLD KMLVD DIGDF TVTN DGATIL SLL DVQHPAG KI LVELAQQQ D REIGDGTTS VVIIASELLK RANELVKNK I HPTTIITG FR VALREAI RFI NEVLST SVDTLGKETL ...文字列: MSQLFNNSRS DTLFLGGEK I SGDDIRNQ NV LATMAVA NVV KSSLGP VGLD KMLVD DIGDF TVTN DGATIL SLL DVQHPAG KI LVELAQQQ D REIGDGTTS VVIIASELLK RANELVKNK I HPTTIITG FR VALREAI RFI NEVLST SVDTLGKETL INIA KTSMS SKIIG ADSD FFSNMV VDA LLAVKTQ NS KGEIKYPV K AVNVLKAHG KSATESLLVP GYALNCTVA S QAMPKRIA GG NVKIACL DLN LQKARM AMGV QINID DPEQL EQIR KREAGI VLE RVKKIID AG AQVVLTTK G IDDLCLKEF VEAKIMGVRR CKKEDLRRI A RATGATLV SS MSNLEGE ETFESSYLGL CDE VVQAKF SDDE CILIK GTSKH SSSS IILRGA NDY SLDEMER SL HDSLSVVK R TLESGNVVP GGGCVEAALN IYLDNFATT V GSREQLAI AE FAAALLI IPK TLAVNA AKDS SELVA KLRSY HAAS QMAKPE DVK RRSYRNY GL DLIRGKIV D EIHAGVLEP TISKVKSLKS ALEACVAIL R IDTMITVD PE PPKEDPH DH |
-分子 #2: T-complex protein 1 subunit beta
分子 | 名称: T-complex protein 1 subunit beta / タイプ: protein_or_peptide / ID: 2 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MSVQIFGDQV TEERAENARL SAFVGAIAVG DLVKSTLGPK GMDKLLQSAS SNTCMVTNDG ATILKSIPLD NPAAKVLVNI SKVQDDEVGD GTTSVTVLSA ELLREAEKLI DQSKIHPQTI IEGYRLASAA ALDALTKAAV DNSHDKTMFR EDLIHIAKTT LSSKILSQDK ...文字列: MSVQIFGDQV TEERAENARL SAFVGAIAVG DLVKSTLGPK GMDKLLQSAS SNTCMVTNDG ATILKSIPLD NPAAKVLVNI SKVQDDEVGD GTTSVTVLSA ELLREAEKLI DQSKIHPQTI IEGYRLASAA ALDALTKAAV DNSHDKTMFR EDLIHIAKTT LSSKILSQDK DHFAELATNA ILRLKGSTNL EHIQIIKILG GKLSDSFLDE GFILAKKFGN NQPKRIENAK ILIANTTLDT DKVKIFGTKF KVDSTAKLAQ LEKAEREKMK NKIAKISKFG INTFINRQLI YDYPEQLFTD LGINSIEHAD FEGVERLALV TGGEVVSTFD EPSKCKLGEC DVIEEIMLGE QPFLKFSGCK AGEACTIVLR GATDQTLDEA ERSLHDALSV LSQTTKETRT VLGGGCAEMV MSKAVDTEAQ NIDGKKSLAV EAFARALRQL PTILADNAGF DSSELVSKLR SSIYNGISTS GLDLNNGTIA DMRQLGIVES YKLKRAVVSS ASEAAEVLLR VDNIIRARPR TANRQHM |
-分子 #3: T-complex protein 1 subunit gamma
分子 | 名称: T-complex protein 1 subunit gamma / タイプ: protein_or_peptide / ID: 3 / 詳細: insertion Strep-CBP-His tag between P374 and K375 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MQAPVVFMNA SQERTTGRQA QISNITAAKA VADVIRTCLG PKAMLKMLLD PMGGLVLTND GHAILREIDV AHPAAKSMLE LSRTQDEEVG DGTTTVIILA GEILAQCAPY LIEKNIHPVI IIQALKKALT DALEVIKQVS KPVDVENDAA MKKLIQASIG TKYVIHWSEK ...文字列: MQAPVVFMNA SQERTTGRQA QISNITAAKA VADVIRTCLG PKAMLKMLLD PMGGLVLTND GHAILREIDV AHPAAKSMLE LSRTQDEEVG DGTTTVIILA GEILAQCAPY LIEKNIHPVI IIQALKKALT DALEVIKQVS KPVDVENDAA MKKLIQASIG TKYVIHWSEK MCELALDAVK TVRKDLGQTV EGEPNFEIDI KRYVRVEKIP GGDVLDSRVL KGVLLNKDVV HPKMSRHIEN PRVVLLDCPL EYKKGESQTN IEIEKEEDWN RILQIEEEQV QLMCEQILAV RPTLVITEKG VSDLAQHYLL KGGCSVLRRV KKSDNNRIAR VTGATIVNRV EDLKESDVGT NCGLFKVEMI GDEYFSFLDN CKEP(LEGSGSGWSHPQFEKGSGKRRWKKNFIAVSAANRFKKISSSGALGSGHHHHHHHHGSGLQ)KACTI MLRGGSKDIL NEIDRNLQDA MAVARNVMLS PSLSPGGGAT EMAVSVKLAE KAKQLEGIQQ WPYQAVADAM ECIPRTLIQN AGGDPIRLLS QLRAKHAQGN FTTGIDGDKG KIVDMVSYGI WEPEVIKQQS VKTAIESACL LLRVDDIVSG VRKQE |
-分子 #4: T-complex protein 1 subunit delta
分子 | 名称: T-complex protein 1 subunit delta / タイプ: protein_or_peptide / ID: 4 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MSAKVPSNAT FKNKEKPQEV RKANIIAARS VADAIRTSLG PKGMDKMIKT SRGEIIISND GHTILKQMAI LHPVARMLVE VSAAQDSEAG DGTTSVVILT GALLGAAERL LNKGIHPTII ADSFQSAAKR SVDILLEMCH KVSLSDREQL VRAASTSLSS KIVSQYSSFL ...文字列: MSAKVPSNAT FKNKEKPQEV RKANIIAARS VADAIRTSLG PKGMDKMIKT SRGEIIISND GHTILKQMAI LHPVARMLVE VSAAQDSEAG DGTTSVVILT GALLGAAERL LNKGIHPTII ADSFQSAAKR SVDILLEMCH KVSLSDREQL VRAASTSLSS KIVSQYSSFL APLAVDSVLK ISDENSKNVD LNDIRLVKKV GGTIDDTEMI DGVVLTQTAI KSAGGPTRKE KAKIGLIQFQ ISPPKPDTEN NIIVNDYRQM DKILKEERAY LLNICKKIKK AKCNVLLIQK SILRDAVNDL ALHFLSKLNI MVVKDIEREE IEFLSKGLGC KPIADIELFT EDRLGSADLV EEIDSDGSKI VRVTGIRNNN ARPTVSVVIR GANNMIIDET ERSLHDALCV IRCLVKERGL IAGGGAPEIE ISRRLSKEAR SMEGVQAFIW QEFASALEVI PTTLAENAGL NSIKVVTELR SKHENGELND GISVRRSGTT NTYEEHILQP VLVSTSAITL ASECVKSILR IDDIAFSR |
-分子 #5: T-complex protein 1 subunit epsilon
分子 | 名称: T-complex protein 1 subunit epsilon / タイプ: protein_or_peptide / ID: 5 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MAARPQQPPM EMPDLSNAIV AQDEMGRPFI IVKDQGNKKR QHGLEAKKSH ILAARSVASI IKTSLGPRGL DKILISPDGE ITITNDGATI LSQMELDNEI AKLLVQLSKS QDDEIGDGTT GVVVLASALL DQALELIQKG IHPIKIANGF DEAAKLAISK LEETCDDISA ...文字列: MAARPQQPPM EMPDLSNAIV AQDEMGRPFI IVKDQGNKKR QHGLEAKKSH ILAARSVASI IKTSLGPRGL DKILISPDGE ITITNDGATI LSQMELDNEI AKLLVQLSKS QDDEIGDGTT GVVVLASALL DQALELIQKG IHPIKIANGF DEAAKLAISK LEETCDDISA SNDELFRDFL LRAAKTSLGS KIVSKDHDRF AEMAVEAVIN VMDKDRKDVD FDLIKMQGRV GGSISDSKLI NGVILDKDFS HPQMPKCVLP KEGSDGVKLA ILTCPFEPPK PKTKHKLDIS SVEEYQKLQT YEQDKFKEMI DDVKKAGADV VICQWGFDDE ANHLLLQNDL PAVRWVGGQE LEHIAISTNG RIVPRFQDLS KDKLGTCSRI YEQEFGTTKD RMLIIEQSKE TKTVTCFVRG SNKMIVDEAE RALHDSLCVV RNLVKDSRVV YGGGAAEVTM SLAVSEEADK QRGIDQYAFR GFAQALDTIP MTLAENSGLD PIGTLSTLKS KQLKEKISNI GVDCLGYGSN DMKELFVVDP FIGKKQQILL ATQLCRMILK IDNVIISGKD EY |
-分子 #6: T-complex protein 1 subunit zeta
分子 | 名称: T-complex protein 1 subunit zeta / タイプ: protein_or_peptide / ID: 6 / 詳細: insertion PA tag within C285 and G286 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MSLQLLNPKA ESLRRDAALK VNVTSAEGLQ SVLETNLGPK GTLKMLVDGA GNIKLTKDGK VLLTEMQIQS PTAVLIARAA AAQDEITGDG TTTVVCLVGE LLRQAHRFIQ EGVHPRIITD GFEIARKESM KFLDEFKISK TNLSNDREFL LQVARSSLLT KVDADLTEVL ...文字列: MSLQLLNPKA ESLRRDAALK VNVTSAEGLQ SVLETNLGPK GTLKMLVDGA GNIKLTKDGK VLLTEMQIQS PTAVLIARAA AAQDEITGDG TTTVVCLVGE LLRQAHRFIQ EGVHPRIITD GFEIARKESM KFLDEFKISK TNLSNDREFL LQVARSSLLT KVDADLTEVL TPIVTDAVLS VYDAQADNLD LHMVEIMQMQ HLSPKDTTFI KGLVLDHGGR HPDMPTRVKN AYVLILNVSL EYEKTEVNSG FFYSSADQRD KLAASERKFV DAKLKKIIDL KNEVC(ggtgtagctatgccaggtgcagaagatgatgtggtg)GMDP DKGFVIINQK GIDPMSLDVF AKHNILALRR AKRRNMERLQ LVTGGEAQNS VEDLSPQILG FSGLVYQETI GEEKFTYVTE NTDPKSCTIL IKGSTHYALA QTKDAVRDGL RAVANVLKDK NIIPGAGAFY IALSRYLRSA NMNKLGAKGK TKTGIEAFAE ALLVIPKTLV KNSGFDPLDV LAMVEDELDD AQDSDETRYV GVDLNIGDSC DPTIEGIWDS YRVLRNAITG ATGIASNLLL CDELLRAGRS TLKETPQ |
-分子 #7: T-complex protein 1 subunit eta
分子 | 名称: T-complex protein 1 subunit eta / タイプ: protein_or_peptide / ID: 7 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MNFGSQTPTI VVLKEGTDAS QGKGQIISNI NACVAVQEAL KPTLGPLGSD ILIVTSNQKT TISNDGATIL KLLDVVHPAA KTLVDISRAQ DAEVGDGTTS VTILAGELMK EAKPFLEEGI SSHLIMKGYR KAVSLAVEKI NELAVDITSE KSSGRELLER CARTAMSSKL ...文字列: MNFGSQTPTI VVLKEGTDAS QGKGQIISNI NACVAVQEAL KPTLGPLGSD ILIVTSNQKT TISNDGATIL KLLDVVHPAA KTLVDISRAQ DAEVGDGTTS VTILAGELMK EAKPFLEEGI SSHLIMKGYR KAVSLAVEKI NELAVDITSE KSSGRELLER CARTAMSSKL IHNNADFFVK MCVDAVLSLD RNDLDDKLIG IKKIPGGAME ESLFINGVAF KKTFSYAGFE QQPKKFNNPK ILSLNVELEL KAEKDNAEVR VEHVEDYQAI VDAEWQLIFE KLRQVEETGA NIVLSKLPIG DLATQFFADR NIFCAGRVSA DDMNRVIQAV GGSIQSTTSD IKPEHLGTCA LFEEMQIGSE RYNLFQGCPQ AKTCTLLLRG GAEQVIAEVE RSLHDAIMIV KRALQNKLIV AGGGATEMEV SKCLRDYSKT IAGKQQMIIN AFAKALEVIP RQLCENAGFD AIEILNKLRL AHSKGEKWYG VVFETENIGD NFAKFVWEPA LVKINALNSA TEATNLILSV DETITNKGSE SANAGMMPPQ GAGRGRGMPM |
-分子 #8: T-complex protein 1 subunit theta
分子 | 名称: T-complex protein 1 subunit theta / タイプ: protein_or_peptide / ID: 8 / 光学異性体: LEVO |
---|---|
由来(天然) | 生物種: ![]() ![]() |
配列 | 文字列: MSLRLPQNPN AGLFKQGYNS YSNADGQIIK SIAAIRELHQ MCLTSMGPCG RNKIIVNHLG KIIITNDAAT MLRELDIVHP AVKVLVMATE QQKIDMGDGT NLVMILAGEL LNVSEKLISM GLSAVEIIQG YNMARKFTLK ELDEMVVGEI TDKNDKNELL KMIKPVISSK ...文字列: MSLRLPQNPN AGLFKQGYNS YSNADGQIIK SIAAIRELHQ MCLTSMGPCG RNKIIVNHLG KIIITNDAAT MLRELDIVHP AVKVLVMATE QQKIDMGDGT NLVMILAGEL LNVSEKLISM GLSAVEIIQG YNMARKFTLK ELDEMVVGEI TDKNDKNELL KMIKPVISSK KYGSEDILSE LVSEAVSHVL PVAQQAGEIP YFNVDSIRVV KIMGGSLSNS TVIKGMVFNR EPEGHVKSLS EDKKHKVAVF TCPLDIANTE TKGTVLLHNA QEMLDFSKGE EKQIDAMMKE IADMGVECIV AGAGVGELAL HYLNRYGILV LKVPSKFELR RLCRVCGATP LPRLGAPTPE ELGLVETVKT MEIGGDRVTV FKQEQGEISR TSTIILRGAT QNNLDDIERA IDDGVAAVKG LMKPSGGKLL PGAGATEIEL ISRITKYGER TPGLLQLAIK QFAVAFEVVP RTLAETAGLD VNEVLPNLYA AHNVTEPGAV KTDHLYKGVD IDGESDEGVK DIREENIYDM LATKKFAINV ATEAATTVLS IDQIIMAKKA GGPRAPQGPR PGNWDQED |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
---|---|
![]() | 単粒子再構成法 |
試料の集合状態 | particle |
-
試料調製
濃度 | 10 mg/mL |
---|---|
緩衝液 | pH: 7.5 |
凍結 | 凍結剤: ETHANE |
-
電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
---|---|
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: SUPER-RESOLUTION / 平均電子線量: 38.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
-
画像解析
最終 再構成 | 解像度のタイプ: BY AUTHOR / 解像度: 11.4 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用した粒子像数: 14805 |
---|---|
初期 角度割当 | タイプ: ANGULAR RECONSTITUTION |
最終 角度割当 | タイプ: ANGULAR RECONSTITUTION |