A guided approach for subtomogram averaging of challenging macromolecular assemblies.
A guided approach for subtomogram averaging of challenging macromolecular assemblies.
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DOI：
10.1016/j.yjsbx.2020.100041
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发表时间：
2020
影响因子：
--
通讯作者：
Grotjahn DA
中科院分区：
文献类型：
--
作者：
Basanta B;Chowdhury S;Lander GC;Grotjahn DA
低温电子显微镜（cryo-EM）是测定大分子配合物三维（3D）结构的有效方法。虽然单粒子电子显微镜因其在解决纯化蛋白质的高分辨率结构方面的应用而广受赞誉，但冷冻电子断层扫描（cryo-ET）已成为在原生或近原生重建细胞环境中可视化大型、瞬态、动态、柔性和/或异质样品结构的领先技术（Baumeister, 2013； Oikonomou和Jensen， 2017）。自动化数据收集（Blocker等人，1997年）软件包（Mastronarde， 2005年，Suloway等人，2009年）和优化的层摄影采集方案（Chreifi等人，2019年，Eisenstein等人，2019年，Hagen等人，2017年，Turoňová等人，2019年）的实施，结合直接电子探测器，能量滤波器和相位板（Khoshouei等人，2017年），彻底改变了可视化细胞机制的可行性，用于功能和生理解释。在重建的层析图中可以识别出感兴趣的生物复合体的多个副本，并且可以在称为子层析图平均（STA）的过程中提取3D“子体积”或“子层析图”并将其平均，以获得感兴趣复合体的更好分辨率的3D重建。已经开发了几种具有不同算法方法的STA处理包，包括PEET (Heumann等人，2011，Nicastro等人，2006),Dynamo （Castano-Diez等人，2017，Castaño-Díez等人，2012）或PyTom （Hrabe等人，2012）。此外，单粒子图像处理的各个方面已被纳入STA处理软件包，如RELION和EMAN2 （Bharat和Scheres， 2016， Bharat等人，2015，Galaz-Montoya等人，2015）。值得注意的是，当结合改进的3d对比度传递函数（CTF）估计和缺失的wedge补偿（Chen等人，2019，Galaz-Montoya等人，2016，Himes和Zhang， 2018， Turoňová等人，2017）时，STA已经实现了亚纳米分辨率下的重建（Himes和Zhang， 2018， Schur等人，2016，Tegunov等人，2020，Turoňová等人，2017），甚至达到了与单粒子分析相当的分辨率。进一步强调了该技术在原位获得高分辨率结构信息方面的前景。然而，尽管仪器和算法有所改进，该领域仍远不能通过STA常规获得高分辨率结构，因为沉积在EM数据库（EMDB）中并通过该方法确定的大多数结构的分辨率低于~ 20 Å（图1a）。此外，虽然在原生原位细胞环境中阐明多型、多亚基复合物结构的能力是cryo-ET和STA相对于其他结构技术的主要优势，但目前的STA处理策略通常仅适用于高度有序、对称、同质的样品，这些样品具有有限的构象变化，并且在单个层析图中存在高拷贝数（图1b）。此类复合物的例子包括纯化病毒和相关病毒复合物（Obr和Schur， 2019），以及高度丰富的细胞质和膜相关核糖体（Orlov等人，2017，Pfeffer等人，2016）。与膜或细丝均匀定向的蛋白质复合物也从该技术中受益匪浅，因为相对高信号的膜或细丝的排列可以帮助驱动噪声较低的感兴趣的低信噪比复合物的初始排列。这些复合物的例子包括轴突动力马达(Grotjahn and Lander…
Cryo-electron microscopy (cryo-EM) is an impactful methodology for three-dimensional (3D) structural determination of macromolecular complexes. While single particle EM gained widespread notoriety for its utility in solving high resolution structures of purified proteins, cryo-electron tomography (cryo-ET) has emerged as the leading technique for visualizing the structures of large, transient, dynamic, flexible, and/or heterogeneous samples in native or near-native reconstituted cellular environments (Baumeister, 2013, Oikonomou and Jensen, 2017). The implementation of automated data collection (Blocker et al., 1997) packages (Mastronarde, 2005, Suloway et al., 2009) and optimized tomographic acquisition schemes (Chreifi et al., 2019, Eisenstein et al., 2019, Hagen et al., 2017, Turoňová et al., 2019), combined with direct electron detectors, energy filters, and phase plates (Khoshouei et al., 2017) has revolutionized the feasibility of visualizing cellular machinery for functional and physiological interpretation. Multiple copies of the biological complex of interest can be identified within reconstructed tomograms, and 3D “subvolumes” or “subtomograms” can be extracted and averaged together in a process called subtomogram averaging (STA) to obtain better-resolved 3D reconstructions of the complex of interest. Several STA processing packages with diverse algorithmic approaches have been developed, including PEET (Heumann et al., 2011, Nicastro et al., 2006), Dynamo (Castano-Diez et al., 2017, Castaño-Díez et al., 2012) or PyTom (Hrabe et al., 2012). Additionally, aspects of single-particle image processing have been incorporated into STA processing packages such as RELION and EMAN2 (Bharat and Scheres, 2016, Bharat et al., 2015, Galaz-Montoya et al., 2015). Notably, when combined with improved 3D-contrast transfer function (CTF) estimation and missing-wedge compensation (Chen et al., 2019, Galaz-Montoya et al., 2016, Himes and Zhang, 2018, Turoňová et al., 2017), STA has been implemented to achieve reconstructions in the sub-nanometer resolution regime (Himes and Zhang, 2018, Schur et al., 2016, Tegunov et al., 2020, Turoňová et al., 2017), even reaching resolutions that are comparable with single particle analyses, further emphasizing the promise of this technique in obtaining high-resolution structural information of complexes in situ.However, despite improvements in instrumentation and algorithms, the field is still far from routinely obtaining high resolution structures by STA, as most structures deposited in the EM Data Bank (EMDB) and determined by this method are at resolutions worse than~ 20 Å (Fig. 1 A). Moreover, while the ability to elucidate the structures of pleomorphic, multi-subunit complexes in native, in situ cellular environment is a major advantage of cryo-ET and STA over other structural techniques, current STA processing strategies are typically only successful for highly ordered, symmetrical, homogenous samples that have limited conformational variations, and are present in high copy numbers within a single tomogram (Fig. 1 B). Examples of such complexes include purified viruses and associated viral complexes (Obr and Schur, 2019), and highly-abundant cytoplasmic and membrane-associated ribosomes (Orlov et al., 2017, Pfeffer et al., 2016). Protein complexes that are uniformly oriented with membranes or filaments have also benefited greatly from this technique, as alignment of the relatively high-signal membrane or filaments can help drive the initial alignment of the noisier, low SNR complex of interest. Examples of these complexes include the axonemal dynein motors (Grotjahn and Lander …
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影响因子：
2.3
作者：
ANDERSEN, AH;KAK, AC
通讯作者：
KAK, AC
影响因子：
3
作者：
Kremer, JR;Mastronarde, DN;McIntosh, JR
通讯作者：
McIntosh, JR
影响因子：
3
作者：
Bruggemann, Jacob;Lander, Gabriel C.;Su, Andrew, I
通讯作者：
Su, Andrew, I
DOI：
10.1083/jcb.137.1.113
发表时间：
1997-04-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Blocker A;Severin FF;Burkhardt JK;Bingham JB;Yu H;Olivo JC;Schroer TA;Hyman AA;Griffiths G
通讯作者：
Griffiths G
影响因子：
3
作者：
Chreifi, Georges;Chen, Songye;Jensen, Grant J.
通讯作者：
Jensen, Grant J.