Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase.
Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase.
复制标题
DOI：
10.1021/acsnano.3c00463
复制
发表时间：
2023-03-28
期刊：
影响因子：
17.1
通讯作者：
Cho, Uhn-Soo
中科院分区：
文献类型：
--
作者：
Ahn, Eungjin;Kim, Byungchul;Park, Soyoung;Erwin, Amanda L.;Sung, Suk Hyun;Hovden, Robert;Mosalaganti, Shyamal;Cho, Uhn-Soo
低温电子显微镜（cryo-EM）已成为一种广泛使用的工具，用于确定蛋白质结构。尽管最近的技术进步，样品制备仍然是一个主要的瓶颈，原因有几个，包括蛋白质变性在空气-水界面，存在的优先取向，不均匀的冰层等。石墨烯，一个二维的碳的同素异形体组成的一个单一的原子层，最近已经获得了关注，作为一个接近理想的支持膜冷冻EM，可以克服这些挑战，因为它的上级属性，包括机械强度和导电性。在这里，我们介绍了一种可靠、易于实施且可重复的方法，可以在1.5天内生产36个石墨烯涂层网格。为了证明其实际应用，我们确定了cryo-EM结构的甲基球菌荚膜可溶性甲烷单加氧酶羟化酶（sMMOH）的分辨率为2.9和2.5微米，分别使用Quantifoil和石墨烯涂层网格。我们发现石墨烯涂层网格具有几个优点，包括所需的蛋白质量较少，并避免了蛋白质在空气-水界面处变性。通过比较的cryo-EM结构的sMMOH与它的晶体结构，我们确定了微妙而显着的几何变化在nonheme二铁中心，这可能更好地表明活性位点的配置sMMOH在休息/氧化状态。
Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air–water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air–water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.
登录
查看更多内容
影响因子：
38.3
作者：
通讯作者：
--
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
7.7
作者：
Kühlbrandt W
通讯作者：
Kühlbrandt W
影响因子：
3
作者：
Han, Bong-Gyoon;Walton, Ross W.;Glaeser, Robert M.
通讯作者：
Glaeser, Robert M.
影响因子：
3
作者：
Kremer, JR;Mastronarde, DN;McIntosh, JR
通讯作者：
McIntosh, JR