ジャーナル: Mol Pharmacol / 年: 2016 タイトル: Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle. 著者: Gabriel A Frank / Suneet Shukla / Prashant Rao / Mario J Borgnia / Alberto Bartesaghi / Alan Merk / Aerfa Mobin / Lothar Esser / Lesley A Earl / Michael M Gottesman / Di Xia / Suresh V ...著者: Gabriel A Frank / Suneet Shukla / Prashant Rao / Mario J Borgnia / Alberto Bartesaghi / Alan Merk / Aerfa Mobin / Lothar Esser / Lesley A Earl / Michael M Gottesman / Di Xia / Suresh V Ambudkar / Sriram Subramaniam / 要旨: The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug ...The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.
ダウンロード / ファイル: emd_3421.map.gz / 形式: CCP4 / 大きさ: 41.9 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Reconstruction of mutant (E556Q/E1201Q) human P-glycoprotein in apo state bound to UIC2 Fab
ボクセルのサイズ
X=Y=Z: 1.403 Å
密度
表面レベル
登録者による: 0.09 / ムービー #1: 0.09
最小 - 最大
-0.02768489 - 0.20235784
平均 (標準偏差)
0.00151615 (±0.01442734)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
224
224
224
Spacing
224
224
224
セル
A=B=C: 314.272 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.403
1.403
1.403
M x/y/z
224
224
224
origin x/y/z
0.000
0.000
0.000
length x/y/z
314.272
314.272
314.272
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-147
-147
-146
NX/NY/NZ
294
294
294
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
224
224
224
D min/max/mean
-0.028
0.202
0.002
-
添付データ
-
試料の構成要素
-
全体 : Human P-glycoprotein (E556Q/E1201Q) in apo state bound to UIC2 Fab
全体
名称: Human P-glycoprotein (E556Q/E1201Q) in apo state bound to UIC2 Fab
要素
試料: Human P-glycoprotein (E556Q/E1201Q) in apo state bound to UIC2 Fab
タンパク質・ペプチド: P-glycoprotein
タンパク質・ペプチド: UIC2 Fab
-
超分子 #1000: Human P-glycoprotein (E556Q/E1201Q) in apo state bound to UIC2 Fab
超分子
名称: Human P-glycoprotein (E556Q/E1201Q) in apo state bound to UIC2 Fab タイプ: sample / ID: 1000 集合状態: One monomer of P-glycoprotein bound to one UIC2 Fab Number unique components: 2
分子量
理論値: 175 KDa
-
分子 #1: P-glycoprotein
分子
名称: P-glycoprotein / タイプ: protein_or_peptide / ID: 1 / Name.synonym: P-gp, Pgp, ABCB1, MDR1 / 詳細: 6-histidine tag at the C-termina / コピー数: 1 / 集合状態: Monomer / 組換発現: Yes
由来(天然)
生物種: Homo sapiens (ヒト) / 別称: Human / 細胞中の位置: Plasma membrane
分子量
理論値: 141 KDa
組換発現
生物種: Trichoplusia ni (イラクサキンウワバ) / 組換株: BTI-TN-5B1-4 / 組換細胞: High Five insect cells
Particles were selected using an automatic selection program. 2D classification was done using EMAN. 3D classification, 3D refinement, and post-processing were done using RELION 1.3.
CTF補正
詳細: CTF parameters obtained from whole micrograph
最終 再構成
想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 12.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: EMAN2, Relion, 1.3 / 詳細: Final maps were calculated from 4 combined datasets / 使用した粒子像数: 34173