+
データを開く
-
基本情報
登録情報 | データベース: PDB / ID: 6ukj | ||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
タイトル | Single-Particle Cryo-EM Structure of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform | ||||||||||||||||||||||||||||||
![]() |
| ||||||||||||||||||||||||||||||
![]() | MEMBRANE PROTEIN / Transporter / drug resistance / digestive vacuole of malaria parasite / nanodisc | ||||||||||||||||||||||||||||||
機能・相同性 | Chloroquine-resistance transporter-like / Chloroquine resistance transporter / CRT-like, chloroquine-resistance transporter-like / vacuolar membrane / xenobiotic transmembrane transporter activity / membrane => GO:0016020 / CHOLESTEROL HEMISUCCINATE / Chloroquine resistance transporter![]() | ||||||||||||||||||||||||||||||
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: 3.3 Å | ||||||||||||||||||||||||||||||
![]() | Kim, J. / Tan, Y.Z. / Wicht, K.J. / Erramilli, S.K. / Dhingra, S.K. / Okombo, J. / Vendome, J. / Hagenah, L.M. / Giacometti, S.I. / Warren, A.L. ...Kim, J. / Tan, Y.Z. / Wicht, K.J. / Erramilli, S.K. / Dhingra, S.K. / Okombo, J. / Vendome, J. / Hagenah, L.M. / Giacometti, S.I. / Warren, A.L. / Nosol, K. / Roepe, P.D. / Potter, C.S. / Carragher, B. / Kossiakoff, A.A. / Quick, M. / Fidock, D.A. / Mancia, F. | ||||||||||||||||||||||||||||||
資金援助 | ![]()
| ||||||||||||||||||||||||||||||
![]() | ![]() タイトル: Structure and drug resistance of the Plasmodium falciparum transporter PfCRT. 著者: Jonathan Kim / Yong Zi Tan / Kathryn J Wicht / Satchal K Erramilli / Satish K Dhingra / John Okombo / Jeremie Vendome / Laura M Hagenah / Sabrina I Giacometti / Audrey L Warren / Kamil Nosol ...著者: Jonathan Kim / Yong Zi Tan / Kathryn J Wicht / Satchal K Erramilli / Satish K Dhingra / John Okombo / Jeremie Vendome / Laura M Hagenah / Sabrina I Giacometti / Audrey L Warren / Kamil Nosol / Paul D Roepe / Clinton S Potter / Bridget Carragher / Anthony A Kossiakoff / Matthias Quick / David A Fidock / Filippo Mancia / ![]() 要旨: The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and ...The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures. | ||||||||||||||||||||||||||||||
履歴 |
|
-
構造の表示
ムービー |
![]() |
---|---|
構造ビューア | 分子: ![]() ![]() |
-
ダウンロードとリンク
-
ダウンロード
PDBx/mmCIF形式 | ![]() | 118.5 KB | 表示 | ![]() |
---|---|---|---|---|
PDB形式 | ![]() | 91 KB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
---|
-関連構造データ
関連構造データ | ![]() 20806MC M: このデータのモデリングに利用したマップデータ C: 同じ文献を引用 ( |
---|---|
類似構造データ | |
電子顕微鏡画像生データ | ![]() Data size: 830.4 Data #1: Unaligned and compressed multi-frame micrographs [micrographs - multiframe] Data #2: Aligned and magnification anisotropy corrected micrographs [micrographs - single frame] Data #3: Final Particle Stack with Refined Euler Angles and Shifts [picked particles - single frame - processed]) |
-
リンク
-
集合体
登録構造単位 | ![]()
|
---|---|
1 |
|
-
要素
#1: タンパク質 | 分子量: 53074.461 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: isolate 7G8 / 遺伝子: PFBG_01689 / 細胞株 (発現宿主): HEK 293 F / 発現宿主: ![]() |
---|---|
#2: 抗体 | 分子量: 25737.631 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() |
#3: 抗体 | 分子量: 23355.898 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() |
#4: 化合物 | ChemComp-Y01 / |
研究の焦点であるリガンドがあるか | Y |
-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
---|---|
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 |
-
試料調製
構成要素 |
| ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
分子量 |
| ||||||||||||||||||||||||
由来(天然) |
| ||||||||||||||||||||||||
由来(組換発現) |
| ||||||||||||||||||||||||
緩衝液 | pH: 7 / 詳細: Solution was filtered and degassed. | ||||||||||||||||||||||||
緩衝液成分 |
| ||||||||||||||||||||||||
試料 | 濃度: 1.56 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: Protein was incorporated into lipid nanodiscs and complexed with Fab. | ||||||||||||||||||||||||
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: UltrAuFoil | ||||||||||||||||||||||||
急速凍結 | 装置: LEICA EM GP / 凍結剤: ETHANE / 湿度: 80 % / 凍結前の試料温度: 279 K / 詳細: Blot for 2 seconds before plunging. |
-
電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
---|---|
顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 215000 X / 最大 デフォーカス(公称値): 1.8 nm / 最小 デフォーカス(公称値): 1.2 nm / Cs: 0.001 mm / アライメント法: OTHER |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 平均露光時間: 6 sec. / 電子線照射量: 91.56 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 撮影したグリッド数: 1 / 実像数: 3377 |
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: 20 eV / 球面収差補正装置: Cs corrector was used. |
画像スキャン | サンプリングサイズ: 5 µm / 横: 3838 / 縦: 3710 / 動画フレーム数/画像: 80 / 利用したフレーム数/画像: 1-80 |
-
解析
EMソフトウェア |
| ||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
画像処理 | 詳細: Energy filter slit width of 20 eV was used during the collection and was aligned automatically every hour using Leginon. | ||||||||||||||||||||||||||||||||||||||||||||||||
CTF補正 | 詳細: CTF was estimated using GCTF and refined throughout the pipeline using cisTEM. タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 183241 | ||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 17030 / アルゴリズム: BACK PROJECTION 詳細: CisTEM was used to reconstruct the final map using original (non-signal subtracted) particles. クラス平均像の数: 1 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: AB INITIO MODEL / 空間: REAL 詳細: Rosetta was used to generate an ab initio model for the PfCRT using the map. 4XMM chains H and L were used as starting point to model the Fab. | ||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 |
|