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データを開く
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基本情報
登録情報 | データベース: PDB / ID: 6v05 | |||||||||||||||
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タイトル | Cryo-EM structure of a substrate-engaged Bam complex | |||||||||||||||
![]() | (Outer membrane protein assembly factor ...) x 6 | |||||||||||||||
![]() | MEMBRANE PROTEIN / beta-barrel / insertase | |||||||||||||||
機能・相同性 | ![]() Bam protein complex / Gram-negative-bacterium-type cell outer membrane assembly / protein insertion into membrane / cell outer membrane / protein-macromolecule adaptor activity / cell adhesion / response to antibiotic / cell surface / identical protein binding / membrane 類似検索 - 分子機能 | |||||||||||||||
生物種 | ![]() ![]() ![]() ![]() | |||||||||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.1 Å | |||||||||||||||
![]() | Tomasek, D. / Rawson, S. / Lee, J. / Wzorek, J.S. / Harrison, S.C. / Li, Z. / Kahne, D. | |||||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structure of a nascent membrane protein as it folds on the BAM complex. 著者: David Tomasek / Shaun Rawson / James Lee / Joseph S Wzorek / Stephen C Harrison / Zongli Li / Daniel Kahne / ![]() 要旨: Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands ...Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP), we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete. | |||||||||||||||
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 335 KB | 表示 | ![]() |
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PDB形式 | ![]() | 264.2 KB | 表示 | ![]() |
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集合体
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要素
-Outer membrane protein assembly factor ... , 6種, 6分子 ABCDEF
#1: タンパク質 | 分子量: 90567.227 Da / 分子数: 1 / 変異: C690S, C700S, F804C / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: K12 / 遺伝子: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / 発現宿主: ![]() ![]() |
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#2: タンパク質 | 分子量: 41918.945 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: K12 / 遺伝子: bamB, yfgL, b2512, JW2496 / 発現宿主: ![]() ![]() |
#3: タンパク質 | 分子量: 36875.277 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: K12 遺伝子: bamC, nlpB, A6581_02455, A6592_19755, A8C65_04235, A8G17_10975, AC067_02730, AC789_1c28090, ACN002_2512, ACN68_11560, ACN81_26800, ACU57_04515, ACU90_01100, AKG99_13055, AM270_06735, AM446_ ...遺伝子: bamC, nlpB, A6581_02455, A6592_19755, A8C65_04235, A8G17_10975, AC067_02730, AC789_1c28090, ACN002_2512, ACN68_11560, ACN81_26800, ACU57_04515, ACU90_01100, AKG99_13055, AM270_06735, AM446_08445, AM464_03885, AML07_10810, AML35_21815, APT94_12605, APZ14_05885, AUQ13_12835, AUS26_08895, AW106_01925, AWP75_05795, B1K96_18050, B9M99_18180, B9T59_21405, BANRA_01021, BANRA_03170, BANRA_04601, BB545_17690, BE963_28130, BEN53_15545, BER14_02350, BHF46_00865, BHS81_15170, BIQ87_14450, BIU72_15910, BIZ41_13405, BK248_16985, BK292_09100, BK373_06140, BK375_00595, BK383_22560, BK400_08740, BMT91_12040, BOH76_02570, BON66_22990, BON69_13590, BON71_05810, BON75_17940, BON76_02345, BON81_08015, BON83_11105, BON86_12870, BON92_23095, BON94_05640, BON95_12010, BON96_16290, BTQ06_24710, BUE81_00175, BvCms2454_00576, BvCms28BK_03651, BvCmsA75A_03498, BvCmsC61A_03920, BvCmsHHP001_02989, BvCmsHHP019_02257, BvCmsHHP056_03727, BvCmsKKP061_00365, BvCmsKSNP019_02678, BvCmsKSNP073_01370, BvCmsKSNP081_01804, BvCmsKSNP120_01449, BvCmsKSP011_03652, BvCmsKSP024_03770, BvCmsKSP026_00127, BvCmsKSP045_01057, BvCmsKSP058_05104, BvCmsKSP067_01143, BvCmsNSNP036_03233, BvCmsNSP007_05045, BvCmsNSP047_01191, BvCmsNSP072_03470, BvCmsSINP011_00589, BvCmsSINP012_02461, BvCmsSINP022_03246, BVL39_14450, BW690_08400, BXT93_09065, BZL31_19830, BZL69_23975, C3449_00575, C4J69_10905, C5N07_10515, C5P01_06205, C5P43_23745, C5P44_22155, C6669_16365, C6986_16055, C7235_07205, C9025_02075, C9083_01895, C9212_02290, C9299_09010, C9E25_13305, C9Y80_06105, C9Y95_21475, C9Z12_16640, CA593_14465, CDL37_24220, CEG98_13220, CG692_24660, CI641_005275, CI694_19990, CIJ94_07570, COD30_00650, COD46_27330, CQP61_08535, CR538_07295, CRM83_27895, CRT46_14950, CWM24_12580, CWS33_10415, CY655_15855, D2184_13985, D2188_18170, D3821_11830, D3822_05395, D3O91_03970, D3Y67_12985, D9D31_00585, D9D43_09275, D9E34_00585, D9E35_17030, D9F17_05285, D9G42_10275, D9G48_19890, D9H68_00610, D9H70_20745, D9H94_18900, D9I11_00510, D9I18_08285, D9I87_03080, D9I88_00655, D9J11_04670, D9J44_00445, D9J60_12530, D9K48_26220, D9K54_08830, DAH27_02200, DAH30_02900, DAH34_02675, DAH37_19960, DBQ99_07990, DD762_13530, DEN86_22815, DEN89_10395, DEN97_02085, DEO19_00630, DIV22_31745, DL545_07645, DL800_18795, DM102_07120, DM129_09435, DNQ41_17525, DNQ45_02690, DP258_07610, DQE83_18935, DQF57_11025, DQO13_11190, DS732_18480, DTL43_01510, DTL90_08635, DTM10_01015, DTM25_19255, DTM45_09650, DU321_10720, DWB25_07365, DXT71_21935, E2112_00485, E2115_16230, E2119_05135, E2126_07380, E2127_04515, E2129_06730, E2132_10070, E2134_09470, E2135_00555, E2855_03225, E2863_03132, E5S46_08270, E5S47_00585, E5S58_10625, E5S61_11100, EAI42_12280, EAI52_02570, EC1094V2_1211, EC3234A_44c00830, EC382_03360, EC95NR1_01696, ECTO6_01409, ED600_00620, ED648_19505, EEP23_23015, EHH55_03705, EIA21_06830, EJC75_11395, EKI52_22355, EL75_1174, EL79_1185, EL80_1190, ELT58_05595, ELU85_09985, ELV08_03665, ELV26_03145, ELV28_11920, EO240_05615, EO241_29005, EPS71_00265, EPS97_10555, EPT01_15160, EQ820_10645, EQ823_08955, EQ825_24025, EQ830_05480, ERL57_11390, ERS085365_02853, ERS085366_00345, ERS085374_00117, ERS085379_00986, ERS085383_02107, ERS085386_00039, ERS085404_01131, ERS085416_01322, ERS139211_02639, ERS150876_00778, ExPECSC038_04470, EXX06_10435, EXX13_08605, EXX23_06350, EXX40_08105, EXX53_08115, EXX55_10135, EXX71_05165, EXX73_17945, EXX78_07240, EXX87_17745, EYD11_06585, EYY78_21115, FAX15_16530, FE198_07360, FNJ69_10855, FNJ79_09165, FNJ83_16390, FORC28_1482, FQR64_06705, FV293_04230, HmCms184_03495, HmCmsJML072_03247, HmCmsJML074_00064, HmCmsJML204_03962, HMPREF3040_02807, HW43_16770, JD73_18025, MJ49_18500, NCTC10090_04434, NCTC10764_00647, NCTC10865_01743, NCTC11022_02534, NCTC11126_06126, NCTC11181_03634, NCTC11341_00816, NCTC12950_01648, NCTC13148_02274, NCTC13462_05111, NCTC13846_01494, NCTC7927_01568, NCTC8621_01448, NCTC8960_04114, NCTC9036_01478, NCTC9044_00198, NCTC9045_01669, NCTC9050_04702, NCTC9055_03350, NCTC9062_01737, NCTC9077_01815, NCTC9111_01883, NCTC9117_01960, NCTC9119_01583, NCTC9702_01656, NCTC9703_01031, NCTC9706_04674, NCTC9969_01610, PGD_00767, RG28_09510, RK56_024525, RX35_01364, SAMEA3472033_00601, SAMEA3472043_00977, SAMEA3472044_02057, SAMEA3472047_03903, SAMEA3472055_00995, SAMEA3472056_00816, SAMEA3472070_00123, SAMEA3472080_01551, SAMEA3472090_01290, SAMEA3472108_03434, SAMEA3472147_00102, SAMEA3484427_00337, SAMEA3484429_00446, SAMEA3484434_00350, SAMEA3485101_04932, SAMEA3485113_01786, SAMEA3752557_01026, SAMEA3752559_00799, SAMEA3752620_00108, SAMEA3753097_02061, SAMEA3753164_00674, SAMEA3753300_00970, SK85_02726, UC41_17115, UN86_00295, UN91_14105, WQ89_05460, WR15_19465, YDC107_1000 発現宿主: ![]() ![]() |
#4: タンパク質 | 分子量: 27858.350 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: K12 / 遺伝子: yfiO, bamD, ECIAI39_2800 / 発現宿主: ![]() ![]() |
#5: タンパク質 | 分子量: 13530.256 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
#6: タンパク質 | 分子量: 64010.285 Da / 分子数: 1 変異: T467C,C690S,C700S,deletion of residues 172-421,deletion of residues 430-439 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: K12 / 遺伝子: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / 発現宿主: ![]() ![]() |
-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: BamABCDE bound to substrate BamA with loop 1 deleted タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT | ||||||||||||||||||||
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分子量 | 実験値: NO | ||||||||||||||||||||
由来(天然) | 生物種: ![]() ![]() | ||||||||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | ||||||||||||||||||||
緩衝液 | pH: 8 | ||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R2/1 | ||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 298 K |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: OTHER / 倍率(補正後): 58717 X / Calibrated defocus min: 1100 nm / 最大 デフォーカス(補正後): 2800 nm |
撮影 | 平均露光時間: 3 sec. / 電子線照射量: 70 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 実像数: 4097 |
電子光学装置 | エネルギーフィルター名称: GIF Bioquantum / エネルギーフィルタースリット幅: 25 eV |
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解析
EMソフトウェア | 名称: SerialEM / カテゴリ: 画像取得 |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION |
粒子像の選択 | 選択した粒子像数: 2054956 |
対称性 | 点対称性: C1 (非対称) |
3次元再構成 | 解像度: 4.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 223353 / 対称性のタイプ: POINT |