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基本情報
登録情報 | データベース: EMDB / ID: EMD-11836 | |||||||||||||||
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タイトル | Cryo-EM density map corresponding to BcsRQAB subcomplex | |||||||||||||||
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機能・相同性 | ![]() bacterial cellulose biosynthetic process / negative regulation of cell division / cytoplasmic side of plasma membrane / cell division / ATP hydrolysis activity / ATP binding / cytosol 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: 4.5 Å | |||||||||||||||
![]() | Zouhir S | |||||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Architecture and regulation of an enterobacterial cellulose secretion system. 著者: Wiem Abidi / Samira Zouhir / Meryem Caleechurn / Stéphane Roche / Petya Violinova Krasteva / ![]() 要旨: Many free-living and pathogenic enterobacteria secrete biofilm-promoting cellulose using a multicomponent, envelope-embedded Bcs secretion system under the control of intracellular second messenger c- ...Many free-living and pathogenic enterobacteria secrete biofilm-promoting cellulose using a multicomponent, envelope-embedded Bcs secretion system under the control of intracellular second messenger c-di-GMP. The molecular understanding of system assembly and cellulose secretion has been largely limited to the crystallographic studies of a distantly homologous BcsAB synthase tandem and a low-resolution reconstruction of an assembled macrocomplex that encompasses most of the inner membrane and cytosolic subunits and features an atypical layered architecture. Here, we present cryo-EM structures of the assembled Bcs macrocomplex, as well as multiple crystallographic snapshots of regulatory Bcs subcomplexes. The structural and functional data uncover the mechanism of asymmetric secretion system assembly and periplasmic crown polymerization and reveal unexpected subunit stoichiometry, multisite c-di-GMP recognition, and ATP-dependent regulation. | |||||||||||||||
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構造の表示
ムービー |
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構造ビューア | EMマップ: ![]() ![]() ![]() |
添付画像 |
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マップデータ | ![]() | 239.2 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 29.2 KB 29.2 KB | 表示 表示 | ![]() |
画像 | ![]() | 45.4 KB | ||
その他 | ![]() ![]() ![]() ![]() | 126.5 MB 131.2 MB 131.4 MB 11.2 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-検証レポート
文書・要旨 | ![]() | 245.7 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 244.9 KB | 表示 | |
XML形式データ | ![]() | 7.1 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
EMDBのページ | ![]() ![]() |
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マップ
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ボクセルのサイズ | X=Y=Z: 1.05248 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
-追加マップ: #1
ファイル | emd_11836_additional_1.map | ||||||||||||
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投影像・断面図 |
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密度ヒストグラム |
-追加マップ: #3
ファイル | emd_11836_additional_2.map | ||||||||||||
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投影像・断面図 |
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密度ヒストグラム |
-追加マップ: #4
ファイル | emd_11836_additional_3.map | ||||||||||||
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投影像・断面図 |
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密度ヒストグラム |
-追加マップ: #2
ファイル | emd_11836_additional_4.map | ||||||||||||
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投影像・断面図 |
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密度ヒストグラム |
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試料の構成要素
-全体 : Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtaine...
全体 | 名称: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc). |
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要素 |
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-超分子 #1: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtaine...
超分子 | 名称: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc). タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all 詳細: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 resin ...詳細: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 resin (Sigma). The proposed stoichiometry for the resultant assembly is BcsR2-Q2-E2-F2-A-B(5-6). Densities corresponding to a BcsR2Q2AB subcomplex were improved after particle subtraction and local refinement in cryoSPARC V2. BcsE and BcsF partake in the assembled Bcs macrocomplex, BcsG does not co-purify stably with the macrocomplex. |
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由来(天然) | 生物種: ![]() ![]() |
組換発現 | 生物種: ![]() ![]() 組換プラスミド: pRSFDuet1-Bcs(Strep)EFG and pCDFDuet1-Bcs(His)RQA(HA-FLAG)B |
分子量 | 理論値: 257 KDa |
-分子 #1: Bacterial cellulose synthase regulator protein BcsB
分子 | 名称: Bacterial cellulose synthase regulator protein BcsB / タイプ: protein_or_peptide / ID: 1 詳細: Genome-encoded BcsB harbors a signal sequence to be addressed to the periplam where according to the ServerP4.1 server prediction would result in a mature form starting with the residue #26 ...詳細: Genome-encoded BcsB harbors a signal sequence to be addressed to the periplam where according to the ServerP4.1 server prediction would result in a mature form starting with the residue #26 TPATQ BcsB sequence was cloned in a pCDFDuet1 vector along Bcs(His)R, BcsQ and BcsA(HA-FLAG). 1 copy in locally refined BcsR2Q2AB map, up to 6 copies in the assembled Bcs macrocomplex. 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() ![]() |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGAD APVV AQNGPSRDVK LTFAQIAPPP GSMVLRGINP NGSIEFGMRS DEVVTKAMLN LEYTPS PSL LPVQSQLKVY LNDELMGVLP VTKEQLGKKT LAQMPINPLF ITDFNRVRLE FVGHYQD VC ENPASTTLWL ...文字列: TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGAD APVV AQNGPSRDVK LTFAQIAPPP GSMVLRGINP NGSIEFGMRS DEVVTKAMLN LEYTPS PSL LPVQSQLKVY LNDELMGVLP VTKEQLGKKT LAQMPINPLF ITDFNRVRLE FVGHYQD VC ENPASTTLWL DVGRSSGLDL TYQTLNVKND LSHFPVPFFD PRDNRTNTLP MVFAGAPD V GLQQASAIVA SWFGSRSGWR GQNFPVLYNQ LPDRNAIVFA TNDKRPDFLR DHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRP VTFGELKTYE EQLQSSGLEP AAINVSLNLP PDLYLMRSTG IDMDINYRYT M PPVKDSSR MDISLNNQFL QSFNLSSKQE ANRLLLRIPV LQGLLDGKTD VSIPALKLGA TN QLRFDFE YMNPMPGGSV DNCITFQPVQ NHVVIGDDST IDFSKYYHFI PMPDLRAFAN AGF PFSRMA DLSQTITVMP KAPNEAQMET LLNTVGFIGA QTGFPAINLT VTDDGSTIQG KDAD IMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGA MAAV IGFQSPYNDQ RSVIALLADS PRGYEMLNDA VNDSGKRATM FGSVAVIRES GINSLR VGD VYYVGHLPWF ERLWYALANH PILLAVLAAI SVILLAWVLW RLLRIISRRR LNPDNE |
-分子 #2: Bacterial cellulose synthase protein BcsA
分子 | 名称: Bacterial cellulose synthase protein BcsA / タイプ: protein_or_peptide / ID: 2 詳細: BcsA sequence was cloned in pCDFDuet1 vector along with Bcs(His)R, BcsQ and BcsB. BcsA harbours a C-terminus HA-Flag Tag. 1 copy in both the locally refined BcsR2Q2AB complex and the assembled Bcs macrocomplex. 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() ![]() |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWLL IGASRKETPK PRRRAFSGLQ NIRGRYHQWM NELPERVSHK TQHLDEKKEL GHLSAGARRL ILGIIVTFSL ILALICVTQP ...文字列: MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWLL IGASRKETPK PRRRAFSGLQ NIRGRYHQWM NELPERVSHK TQHLDEKKEL GHLSAGARRL ILGIIVTFSL ILALICVTQP FNPLAQFIFL MLLWGVALIV RRMPGRFSAL MLIVLSLTVS CRYIWWRYTS TLNWDDPVSL VCGLILLFAE TYAWIVLVLG YFQVVWPLNR QPVPLPKDMS LWPSVDIFVP TYNEDLNVVK NTIYASLGID WPKDKLNIWI LDDGGREEFR QFAQNVGVKY IARTTHEHAK AGNINNALKY AKGEFVSIFD CDHVPTRSFL QMTMGWFLKE KQLAMMQTPH HFFSPDPFER NLGRFRKTPN EGTLFYGLVQ DGNDMWDATF FCGSCAVIRR KPLDEIGGIA VETVTEDAHT SLRLHRRGYT SAYMRIPQAA GLATESLSAH IGQRIRWARG MVQIFRLDNP LTGKGLKFAQ RLCYVNAMFH FLSGIPRLIF LTAPLAFLLL HAYIIYAPAL MIALFVLPHM IHASLTNSKI QGKYRHSFWS EIYETVLAWY IAPPTLVALI NPHKGKFNVT AKGGLVEEEY VDWVISRPYI FLVLLNLVGV AVGIWRYFYG PPTEMLTVVV SMVWVFYNLI VLGGAVAVSV ESKQVRRSHR VEMTMPAAIA REDGHLFSCT VQDFSDGGLG IKINGQAQIL EGQKVNLLLK RGQQEYVFPT QVARVMGNEV GLKLMPLTTQ QHIDFVQCTF ARADTWALWQ DSYPEDKPLE SLLDILKLGF RGYRHLAEFA PSSVKGIFRV LTSLVSWVVS FIPRRPERSE TAQPSDQALA QQGSARSSGR TGLEFEEFYP YDVPDYAADY KDDDDKRS |
-分子 #3: Bacterial cellulose synthase regulator protein BcsR
分子 | 名称: Bacterial cellulose synthase regulator protein BcsR / タイプ: protein_or_peptide / ID: 3 詳細: BcsR sequence was cloned in pCDFDuet1 vector along with BcsQ, BcsA(HA-FLAG) and BcsB. BcsR harbours a N-terminal octahistidine tag (N-His8). Likely two copies in the assembled secretion ...詳細: BcsR sequence was cloned in pCDFDuet1 vector along with BcsQ, BcsA(HA-FLAG) and BcsB. BcsR harbours a N-terminal octahistidine tag (N-His8). Likely two copies in the assembled secretion complexes, however the local resolution does not allow backbone tracing. High-resolution structures solved by X-ray crystallography. 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() ![]() |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MGSSHHHHHH HHAAGSMNNN EPDTLPDPAI GYIFQNDIVA LKQAFSLPDI DYADISQREQ LAAALKRWPL LAEFAQQK |
-分子 #4: Bacterial cellulose synthase regulator protein BcsQ
分子 | 名称: Bacterial cellulose synthase regulator protein BcsQ / タイプ: protein_or_peptide / ID: 4 詳細: BcsQ sequence was cloned in pCDFDuet1 vector along with Bcs(His)R, BcsA(HA-FLAG) and BcsB 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() ![]() |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI ...文字列: MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI NNFRIGSQVQ DDIYQLWLQS QRRLLPMLIH RDEAMAECLA AKQPVGEYRS DALAAEEILT LANWCLLNYS GLKTPVGSKS |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
濃度 | 0.8 mg/mL | ||||||||||||||
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緩衝液 | pH: 8 構成要素:
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グリッド | モデル: Quantifoil R1.2/1.3 / 材質: GOLD / 前処理 - タイプ: GLOW DISCHARGE / 詳細: Elmo Glow Discharge system | ||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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特殊光学系 | エネルギーフィルター - 名称: GIF Quantum LS |
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / 平均電子線量: 1.2 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | C2レンズ絞り径: 100.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): 2.75 µm / 最小 デフォーカス(公称値): 0.75 µm / 倍率(公称値): 130000 |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |