+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-27149 | ||||||||||||
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タイトル | Zebrafish MFSD2A isoform B in inward open ligand-free conformation | ||||||||||||
マップデータ | Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution. | ||||||||||||
試料 |
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機能・相同性 | 機能・相同性情報 Synthesis of PC / fatty acid transmembrane transporter activity / lysophospholipid translocation / lysophospholipid:sodium symporter activity / lysophospholipid transport / lipid transport across blood-brain barrier / establishment of blood-brain barrier / symporter activity / transcytosis / carbohydrate transport ...Synthesis of PC / fatty acid transmembrane transporter activity / lysophospholipid translocation / lysophospholipid:sodium symporter activity / lysophospholipid transport / lipid transport across blood-brain barrier / establishment of blood-brain barrier / symporter activity / transcytosis / carbohydrate transport / fatty acid transport / endoplasmic reticulum membrane / plasma membrane 類似検索 - 分子機能 | ||||||||||||
生物種 | Danio rerio (ゼブラフィッシュ) / Mus musculus (ハツカネズミ) | ||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.4 Å | ||||||||||||
データ登録者 | Nguyen C / Lei HT / Lai LTF / Gallentino MJ / Mu X / Matthies D / Gonen T | ||||||||||||
資金援助 | 米国, 3件
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引用 | ジャーナル: Nat Commun / 年: 2023 タイトル: Lipid flipping in the omega-3 fatty-acid transporter. 著者: Chi Nguyen / Hsiang-Ting Lei / Louis Tung Faat Lai / Marc J Gallenito / Xuelang Mu / Doreen Matthies / Tamir Gonen / 要旨: Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions ...Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the recently determined structures of Mfsd2a, the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease. | ||||||||||||
履歴 |
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-構造の表示
添付画像 |
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-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_27149.map.gz | 5.7 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-27149-v30.xml emd-27149.xml | 33.6 KB 33.6 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_27149_fsc.xml | 12.7 KB | 表示 | FSCデータファイル |
画像 | emd_27149.png | 59.2 KB | ||
その他 | emd_27149_additional_1.map.gz emd_27149_additional_2.map.gz emd_27149_half_map_1.map.gz emd_27149_half_map_2.map.gz | 107.8 MB 204.1 MB 200.8 MB 200.8 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-27149 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27149 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_27149_validation.pdf.gz | 686.8 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_27149_full_validation.pdf.gz | 686.4 KB | 表示 | |
XML形式データ | emd_27149_validation.xml.gz | 21.7 KB | 表示 | |
CIF形式データ | emd_27149_validation.cif.gz | 27.9 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27149 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27149 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_27149.map.gz / 形式: CCP4 / 大きさ: 216 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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注釈 | Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution. | ||||||||||||||||||||||||||||||||||||
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: 0.844 Å | ||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-追加マップ: Single-particle cryo-EM map of MFSD2A isoform B from...
ファイル | emd_27149_additional_1.map | ||||||||||||
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注釈 | Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, unsharpened. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-追加マップ: Single-particle cryo-EM map of MFSD2A isoform B from...
ファイル | emd_27149_additional_2.map | ||||||||||||
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注釈 | Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, sharpened B-114. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Single-particle cryo-EM half map A of MFSD2A isoform...
ファイル | emd_27149_half_map_1.map | ||||||||||||
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注釈 | Single-particle cryo-EM half map A of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Single-particle cryo-EM half map B of MFSD2A isoform...
ファイル | emd_27149_half_map_2.map | ||||||||||||
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注釈 | Single-particle cryo-EM half map B of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-試料の構成要素
-全体 : Single-particle cryo-EM map of MFSD2A isoform B from zebrafish wi...
全体 | 名称: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution |
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要素 |
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-超分子 #1: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish wi...
超分子 | 名称: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution タイプ: complex / ID: 1 / キメラ: Yes / 親要素: 0 / 含まれる分子: #1-#3 |
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由来(天然) | 生物種: Danio rerio (ゼブラフィッシュ) |
分子量 | 理論値: 56 KDa |
-分子 #1: Sodium-dependent lysophosphatidylcholine symporter 1-B
分子 | 名称: Sodium-dependent lysophosphatidylcholine symporter 1-B タイプ: protein_or_peptide / ID: 1 / コピー数: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: Danio rerio (ゼブラフィッシュ) |
分子量 | 理論値: 56.683965 KDa |
組換発現 | 生物種: Spodoptera frugiperda (ツマジロクサヨトウ) |
配列 | 文字列: MHHHHHHHHH HENLYFQGGS DEVKLAKHET KSRLSVCSKL CYAIGGAPYQ ITGCAIGFFL QIYLLDVALL DPFYASIILF VGRAWDAVT DPTVGFLVSR TPWTRFGRMM PWIVLSTPFA VLCYFLIWYV PSVDQGKVVW YLIFYCCFQT LQTCFHVPYS A LTMFISTE ...文字列: MHHHHHHHHH HENLYFQGGS DEVKLAKHET KSRLSVCSKL CYAIGGAPYQ ITGCAIGFFL QIYLLDVALL DPFYASIILF VGRAWDAVT DPTVGFLVSR TPWTRFGRMM PWIVLSTPFA VLCYFLIWYV PSVDQGKVVW YLIFYCCFQT LQTCFHVPYS A LTMFISTE QKERDSATAY RMTVEVLGTL IGTAIQGQIV GMANAPCIST EIDLQSTGLE VAPDVQITDP HVSLQDLRNA YM IASGVIC AIYVVCAVVL FLGVKEQKDT CRVRTEPMSF FQGICMVMGH GPYAKLVMGF LFTSLAFMLL EGNFALFCIY NLG FRNDFQ NVLLVIMLSA TLAIPFWQWF LTKFGKKTAV YIGTTSVVPF LISVVLVPSS LAVTYIASFA AGVSVAAAFL LPWS MLPDV VDDFKVQNPE SQGHEAIFYS FYVFFTKFAS GVSLGVSTLS LDFAGYVTRG CTQPGEVKLT LKILVSAAPI VLIII GLLI FISYPINEEK RQGNRKLLNE QREQ |
-分子 #2: FAB light chain
分子 | 名称: FAB light chain / タイプ: protein_or_peptide / ID: 2 / コピー数: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: Mus musculus (ハツカネズミ) |
分子量 | 理論値: 20.674949 KDa |
組換発現 | 生物種: Mus musculus (ハツカネズミ) |
配列 | 文字列: ALDINSPEAE KNAKGARARI TCNAGNQVGS AVAWFNQRPG DPASLLTYWA ATEKGVAGKQ SAQGASTKFS MSSAGPEAPS LSSYWCLLF EKGAFSFGGS KLNPREGAGP QASILPPSAD LNTSGGAAVV CFLPNWYGNI TVQWKTEAPQ SQANMSWPGQ A GANAAYAM ...文字列: ALDINSPEAE KNAKGARARI TCNAGNQVGS AVAWFNQRPG DPASLLTYWA ATEKGVAGKQ SAQGASTKFS MSSAGPEAPS LSSYWCLLF EKGAFSFGGS KLNPREGAGP QASILPPSAD LNTSGGAAVV CFLPNWYGNI TVQWKTEAPQ SQANMSWPGQ A GANAAYAM AAVLAITKGD YGPGSFTCNA SNRGTGPFAM SLN |
-分子 #3: FAB heavy chain
分子 | 名称: FAB heavy chain / タイプ: protein_or_peptide / ID: 3 / コピー数: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: Mus musculus (ハツカネズミ) |
分子量 | 理論値: 20.568018 KDa |
組換発現 | 生物種: Mus musculus (ハツカネズミ) |
配列 | 文字列: ASKLELSGPA EPRGSKSAQI TCKAKGFPEA RFWVFWLFQR AAALDWPAAN FSGGPVQFES RFQGNASLKG SQAQANAELN IGALGSSTA TYRCGWKLAN GGFFPSWGGA NVNGAAGAKA PAVYPVEISG AGTGSVTLGC LVKGYNAKPN LTWPGASGAL T FPSELNGA ...文字列: ASKLELSGPA EPRGSKSAQI TCKAKGFPEA RFWVFWLFQR AAALDWPAAN FSGGPVQFES RFQGNASLKG SQAQANAELN IGALGSSTA TYRCGWKLAN GGFFPSWGGA NVNGAAGAKA PAVYPVEISG AGTGSVTLGC LVKGYNAKPN LTWPGASGAL T FPSELNGA LWNLASAVTG SGFPSATCAV GFGAATDVDK KVAAA |
-分子 #4: DODECYL-BETA-D-MALTOSIDE
分子 | 名称: DODECYL-BETA-D-MALTOSIDE / タイプ: ligand / ID: 4 / コピー数: 10 / 式: LMT |
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分子量 | 理論値: 510.615 Da |
Chemical component information | ChemComp-LMT: |
-分子 #5: water
分子 | 名称: water / タイプ: ligand / ID: 5 / コピー数: 4 / 式: HOH |
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分子量 | 理論値: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-試料調製
濃度 | 3 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: COPPER / メッシュ: 400 / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 120 sec. / 前処理 - 雰囲気: AIR | ||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 86 % / チャンバー内温度: 277 K / 装置: LEICA EM GP 詳細: 400-mesh 1.2/1.3 Cu grids (Quantifoil) were made hydrophilic by glow discharging for two times 60 seconds with a current of 15 mA in a PELCO easiGlow system. The cryo grids were produced ...詳細: 400-mesh 1.2/1.3 Cu grids (Quantifoil) were made hydrophilic by glow discharging for two times 60 seconds with a current of 15 mA in a PELCO easiGlow system. The cryo grids were produced using a Leica EM GP (Leica). The chamber was kept at 4 C and 95% humidity (86-91% measured). 3 microliter sample at 3 mg/ml was applied to a glow-discharged holey grid, blotted for 6 s, and plunge frozen into liquid ethane and stored in liquid nitrogen.. | ||||||||
詳細 | Purified 15A9 FAB fragment was incubated overnight at 4 C with MFSD2A at a 5:1 molar ratio. DrMFSD2A-15A9 complex was injected into the size exclusion column Superdex200 to separate the unbound FAB. The size exclusion column step was also used to exchange the buffer of the MFSD2A-FAB complex into 50 mM HEPES, 150 mM NaCl and 0.02% DDM. SDS-PAGE was used to pool peak fractions containing both MFSD2A and FAB, indicating complex formation. The pooled fractions containing MFSD2A-FAB were concentrated to 3 mg/ml using Amicon spin concentrator with a 30 kDa cutoff. |
-電子顕微鏡法
顕微鏡 | TFS KRIOS |
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特殊光学系 | 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: 20 eV |
詳細 | Cryo-EM grids were loaded into a 300 keV FEI Titan Krios cryo electron microscope (ThermoFisher Scientific, formerly FEI) at HHMI Janelia Reasearch Campus, Janelia Krios 1, equipped with a Cs corrector, and Gatan energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with 1 e-/A2 per frame (50 e-/A2 total dose) were automatically recorded at a nominal magnification of 81,000x, corresponding to a physical pixel size of 0.844 A/px (superresolution pixel size 0.422 A/px) in CDS mode at a dose rate of 9.5 e-/px/s (~7.5 e-/px/s on the camera through the sample) and a defocus range of -0.5 to -1.8 micrometer using SerialEM. In total, 2,653 and 8,443 movies were collected in two separate imaging sessions, with the first dataset on a 400-mesh copper grid and the second on a 300-mesh UltrAuFoil gold grid. |
撮影 | フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 撮影したグリッド数: 2 / 実像数: 11096 / 平均露光時間: 3.75 sec. / 平均電子線量: 50.0 e/Å2 詳細: Movies of 50 frames with 1 e-/A2 per frame (50 e-/A2 total dose) were automatically recorded at a nominal magnification of 81,000x, corresponding to a physical pixel size of 0.844 A/px ...詳細: Movies of 50 frames with 1 e-/A2 per frame (50 e-/A2 total dose) were automatically recorded at a nominal magnification of 81,000x, corresponding to a physical pixel size of 0.844 A/px (superresolution pixel size 0.422 A/px) in CDS mode at a dose rate of 9.5 e-/px/s (~7.5 e-/px/s on the camera through the sample) and a defocus range of -0.5 to -1.8 mircometer using SerialEM. In total, 2,653 and 8,443 movies were collected in two separate imaging sessions, with the first dataset on a 400-mesh copper grid and the second on a 300-mesh UltrAuFoil gold grid. |
電子線 | 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN |
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: 0.01 mm / 最大 デフォーカス(公称値): 1.8 µm / 最小 デフォーカス(公称値): 0.5 µm / 倍率(公称値): 81000 |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
+画像解析
-原子モデル構築 1
初期モデル | PDB ID: |
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精密化 | 空間: REAL / プロトコル: RIGID BODY FIT |
得られたモデル | PDB-8d2t: |