+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 5sv9 | |||||||||||||||
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タイトル | Structure of the SLC4 transporter Bor1p in an inward-facing conformation | |||||||||||||||
要素 | Bor1p boron transporter | |||||||||||||||
キーワード | TRANSPORT PROTEIN / boron transporter / anion exchanger family / alternating access mechanism / Structural Genomics / PSI-Biology / Transcontinental EM Initiative for Membrane Protein Structure / TEMIMPS | |||||||||||||||
機能・相同性 | Bicarbonate transporter, eukaryotic / Bicarbonate transporter-like, transmembrane domain / HCO3- transporter integral membrane domain / solute:inorganic anion antiporter activity / membrane => GO:0016020 / Bor1p boron transporter 機能・相同性情報 | |||||||||||||||
生物種 | Saccharomyces mikatae (酵母) | |||||||||||||||
手法 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 5.9 Å | |||||||||||||||
データ登録者 | Coudray, N. / Seyler, S. / Lasala, R. / Zhang, Z. / Clark, K.M. / Dumont, M.E. / Rohou, A. / Beckstein, O. / Stokes, D.L. / Transcontinental EM Initiative for Membrane Protein Structure (TEMIMPS) | |||||||||||||||
資金援助 | 米国, 4件
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引用 | ジャーナル: Protein Sci / 年: 2017 タイトル: Structure of the SLC4 transporter Bor1p in an inward-facing conformation. 著者: Nicolas Coudray / Sean L Seyler / Ralph Lasala / Zhening Zhang / Kathy M Clark / Mark E Dumont / Alexis Rohou / Oliver Beckstein / David L Stokes / 要旨: Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate ...Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily. | |||||||||||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 5sv9.cif.gz | 176.6 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb5sv9.ent.gz | 132.4 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 5sv9.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 5sv9_validation.pdf.gz | 742.3 KB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 5sv9_full_validation.pdf.gz | 854.9 KB | 表示 | |
XML形式データ | 5sv9_validation.xml.gz | 46.6 KB | 表示 | |
CIF形式データ | 5sv9_validation.cif.gz | 66.3 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/sv/5sv9 ftp://data.pdbj.org/pub/pdb/validation_reports/sv/5sv9 | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
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1 |
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対称性 | らせん対称: (回転対称性: 1 / Dyad axis: yes / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 4.8 Å / Rotation per n subunits: 37.35 °) |
-要素
#1: タンパク質 | 分子量: 53829.766 Da / 分子数: 2 / 由来タイプ: 組換発現 / 由来: (組換発現) Saccharomyces mikatae (酵母) / 発現宿主: Saccharomyces cerevisiae (パン酵母) / 参照: UniProt: A0A1C7D1B8*PLUS |
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-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: HELICAL ARRAY / 3次元再構成法: らせん対称体再構成法 |
-試料調製
構成要素 | 名称: Bor1p dimer in an inward-facing conformation / タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT | |||||||||||||||||||||||||
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分子量 | 値: 0.06 MDa / 実験値: NO | |||||||||||||||||||||||||
由来(天然) | 生物種: Saccharomyces mikatae (酵母) | |||||||||||||||||||||||||
由来(組換発現) | 生物種: Saccharomyces cerevisiae (パン酵母) / プラスミド: pSGP46 | |||||||||||||||||||||||||
緩衝液 | pH: 7 / 詳細: Buffer was changed twice per day. | |||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.25 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: The protein was solubilized in 1% n-Dodecyl beta-D-maltoside and exchanged into heptaethyleneglycol-n-dodecylether (C12E7) while bound to the IgG Sepharose affinity column. | |||||||||||||||||||||||||
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: EMS | |||||||||||||||||||||||||
急速凍結 | 装置: HOMEMADE PLUNGER / 凍結剤: ETHANE / 湿度: 90 % |
-電子顕微鏡撮影
実験機器 | モデル: Tecnai F20 / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TECNAI F20 |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 200 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 19000 X / 最大 デフォーカス(公称値): 2500 nm / 最小 デフォーカス(公称値): 1000 nm / Calibrated defocus min: 850 nm / 最大 デフォーカス(補正後): 2700 nm / Cs: 2 mm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER |
撮影 | 平均露光時間: 6.75 sec. / 電子線照射量: 45 e/Å2 / 検出モード: SUPER-RESOLUTION フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 撮影したグリッド数: 2 / 実像数: 252 |
画像スキャン | 横: 3710 / 縦: 3710 / 動画フレーム数/画像: 27 / 利用したフレーム数/画像: 2-27 |
-解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
らせん対称 | 回転角度/サブユニット: 37.35 ° / 軸方向距離/サブユニット: 4.8 Å / らせん対称軸の対称性: C1 | ||||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 87 | ||||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 5.9 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 75 詳細: Reconstruction was done using D1 symmetry because of an existing two-fold symmetry in the unit cells composing the helical lattice. This two-fold axis runs perpendicular to the helical axis. ...詳細: Reconstruction was done using D1 symmetry because of an existing two-fold symmetry in the unit cells composing the helical lattice. This two-fold axis runs perpendicular to the helical axis. For the final structure, a filter was applied to the map in order to compensate for resolution-dependent amplitude falloff. To do so, we built a model by arranging UraA in a helical assembly in order to mimic the mass distribution in Bor1p tubes. Fourier transforms from this model and from the experimental maps were then rotationally averaged to produce 1D scattering profiles. The resolution-dependent amplitude ratio from these profiles was used as a filter that was applied to the experimental amplitudes using SPARX routines. Finally, a low-pass filter was applied with a 5 Angstrom stop-band frequency. 対称性のタイプ: HELICAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: cross-correlation coefficient 詳細: A homology model was first created using human AE1 (PDB entry 4YZF) as a template using MODELLER, placed into the density map using SITUS, and then fitted into the map using the Molecular ...詳細: A homology model was first created using human AE1 (PDB entry 4YZF) as a template using MODELLER, placed into the density map using SITUS, and then fitted into the map using the Molecular Dynamics Flexible Fitting (MDFF) method. | ||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | 3D fitting-ID: 1 / Accession code: 4YZF / Initial refinement model-ID: 1 / Pdb chain residue range: 418-911 / PDB-ID: 4YZF / Source name: PDB / タイプ: experimental model
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