ジャーナル: Nat Commun / 年: 2020 タイトル: Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins. 著者: Somnath Mukherjee / Satchal K Erramilli / Mark Ammirati / Frances J D Alvarez / Kimberly F Fennell / Michael D Purdy / Blazej M Skrobek / Katarzyna Radziwon / John Coukos / Yanyong Kang / ...著者: Somnath Mukherjee / Satchal K Erramilli / Mark Ammirati / Frances J D Alvarez / Kimberly F Fennell / Michael D Purdy / Blazej M Skrobek / Katarzyna Radziwon / John Coukos / Yanyong Kang / Przemysław Dutka / Xiang Gao / Xiayang Qiu / Mark Yeager / H Eric Xu / Seungil Han / Anthony A Kossiakoff / 要旨: We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an ...We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.
名称: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline タイプ: COMPLEX / Entity ID: #1-#4 / 由来: RECOMBINANT
分子量
実験値: NO
由来(天然)
生物種: Homo sapiens (ヒト)
由来(組換発現)
生物種: Homo sapiens (ヒト)
緩衝液
pH: 7.5
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
20mM
Tris
C4H11NO3
1
2
150mM
sodiumchloride
NaCl
1
3
0.1mM
tris(2-carboxyethyl)phosphine
C9H15O6P
1
4
100uM
varenicline
C13H13N3
1
5
0.018 %
n-dodecyl-beta-D-maltoside
C24H46O1
1
6
0.006 %
CholesterylHemisuccinate
C31H50O4
1
試料
濃度: 3 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES