例如可是尝试在不同气体环境:比如空气或在正戊胺(N-amylamine)(Nguyen et al., 2015)环境下做辉光放电,或氢气、氧气等环境做等离子清洗。不同气体解离形成不同电荷,比如H2解离带形成H+带正电,O2解离形成O2-带负电等,这样可以改变载网亲水处理之后的表面性质,从而改变与蛋白质的相互作用,最终使得被成功吸附的蛋白分子取向分布出现显著变化。
当然这个是基于上条的暴力破解。简单说就是大量2D分类之后,把主要的优势取向混合,然后随机扔掉大部分颗粒。这个过程具体扔掉多少要看取向有多严重,以及自己有多少颗粒(百万级土豪随意)。具体操作,可以通过尝试法不同比例看那个结果最好,比方95%,90%,85%,80%,70%,60%,50%都试一下,只要有足够的计算资源,这是最有效的捷径。这个过程可以应用到3D,也可以迭代,也即等有了好的reference之后,取向也算的更加准确,这个时候把以前扔掉的颗粒再重新捡回来处理,再扔,再处理,直到满意为止。具体细节可参照dynactin(Urnavicius et al., 2015)和动力蛋白dynein(Zhang et al 2017)冷冻电镜数据处理,更多细节可email:kzhang@mrc-lmb.cam.ac.uk
Boland, A., Martin,T.G., Zhang, Z., Yang, J., Bai, X.C., Chang, L., Scheres, S.H., and Barford, D.(2017). Cryo-EM structure of a metazoan separase-securin complex at near-atomicresolution. Nat Struct Mol Biol 24,414-418.
Lander, G.C., Estrin, E.,Matyskiela, M.E., Bashore, C., Nogales, E., and Martin, A. (2012). Completesubunit architecture of the proteasome regulatory particle. Nature 482, 186-191.
Nguyen, T.H., Galej, W.P., Bai,X.C., Savva, C.G., Newman, A.J., Scheres, S.H., and Nagai, K. (2015). Thearchitecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47-52.
Russo, C.J., and Passmore, L.A.(2014). Electron microscopy: Ultrastable gold substrates for electroncryomicroscopy. Science 346,1377-1380.
Russo, C.J., and Passmore, L.A.(2016). Ultrastable gold substrates: Properties of a support forhigh-resolution electron cryomicroscopy of biological specimens. J Struct Biol 193, 33-44.
Urnavicius, L.*, Zhang, K.*,Diamant, A.G., Motz, C., Schlager, M.A., Yu, M., Patel, N.A., Robinson, C.V.,and Carter, A.P. (2015). The structure of the dynactin complex and itsinteraction with dynein. Science 347,1441-1446.
K. Zhang*, H.E. Foster* et al. (2017).“Cryo-EM Reveals How Human Cytoplasmic Dynein IsAuto-inhibited and Activated.”Cell, 169, 1303–1314.http://www.cell.com/cell/fulltext/S0092-8674(17)30585-8
Yu, G., Li, K., Huang, P., Jiang,X., and Jiang, W. (2016). Antibody-Based Affinity Cryoelectron Microscopy at2.6-A Resolution. Structure 24,1984-1990.
Yu, G., Vago, F., Zhang, D.,Snyder, J.E., Yan, R., Zhang, C., Benjamin, C., Jiang, X., Kuhn, R.J., Serwer, P., et al. (2014). Single-stepantibody-based affinity cryo-electron microscopy for imaging and structuralanalysis of macromolecular assemblies. J Struct Biol 187, 1-9.
Zang, Y., Jin, M., Wang, H., Cui,Z., Kong, L., Liu, C., and Cong, Y. (2016). Staggered ATP binding mechanism ofeukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM. NatStruct Mol Biol 23, 1083-1091.
Zhang, K. (2016). Gctf: Real-timeCTF determination and correction. J Struct Biol 193, 1-12.