ELECTRON DIFFRACTION OF MITOCHONDRIAL MEMBRANE PROTEINS

(支持NSF MCB 9506113至C.A.Mannella)VDAC占主导地位 线粒体外膜上的通道，由单一拷贝形成 一种30kD的蛋白质。有相当多的证据表明，这条海峡是一条 β桶状细菌孔蛋白，但与孔蛋白不同的是，这个通道 具有多个刺激的门(打开和关闭)，包括低幅度 跨膜电位和pH值在5以下。门控机制是 未知且具有相当大的兴趣。我们已经能够发展壮大 该蛋白质的柱状2D晶体通过处理外膜 粗面核线粒体内含磷脂酶A2。使用低剂量电子 显微镜和图像处理，我们已经生成了低分辨率的 (1.7 nm)硫代葡萄糖胞内通道的三维结构。我们想要 通过使用电子将这种结构扩展到更高的分辨率 冷冻水化样品的衍射法采集傅里叶振幅 信息，这些信息将通过低分辨率图像和相位进行分阶段 延期程序 与道格·多塞特合作进行锻炼 (豪普特曼-伍德沃德基金会，纽约州布法罗)。圆柱形的 VDAC的晶体很小，从两层膜反射 这些层经常重叠，这限制了这些晶体的用途。 用于电子衍射(ED)实验。然而，我们发现， 将这些囊泡打开成扁平的单层薄片的方法 接近1微米的线性尺寸。我们将确定 冷冻水合状态下的ED数据的质量和 评估它们是否适合认真尝试3D数据 收集。在100、400和1000千伏下收集的ED数据的质量 将进行比较，以确定直接分阶段方法是否 可行性，即Ewald球体是否有预期的改进 扁平化和减少的动态散射使显著的 尝试将直接阶段化方法应用于 蛋白质结构测定。线粒体外膜是 分离，VDAC晶体是通过去除一些 使用磷脂酶A的膜脂(聚乳酸，来自蜂窝静脉)。这个 膜晶体由泡状变为扁平。 通过使用磷酸盐、柠檬酸盐和 甘氨酸缓冲液。酶水平较高的馏分形成晶体 首先，从酶含量较低的馏分中提取晶体 随后几天的水平。晶体必须是负色的，否则 在EM网格形成后立即跳跃-冻结，否则剩余 酵素会摧毁它们。缓冲液和酶的测试 稳定片状晶体的抑制剂正在进行中。 泡状和片状的晶体首先用 在CTEM和HVEM上进行阴性染色，然后在CTEM上使用冷冻EM。 当结果看起来很有希望时，将使用冷冻-EM进行比较 HVEM和IVEM。给出了样品的衍射和成像条件。 为CTEM和HVEM设计了晶体，以及 在负染晶体上观察到了衍射斑。这个 第一项低温电磁工作现在正在进行中。

(Support NSF MCB 9506113 to C.A. Mannella) VDAC is the predominant channel in the outer membrane of mitochondria, formed by a single copy of a 30-KD protein. There is considerable evidence the channel is a beta-barrel-like bacterial porin but, unlike the porins, this channel gates (open and closes) with several stimuli, including low-amplitude transmembrane potential and pH below 5. The mechanism of gating is unknown and of considerable interest. We have been able to grow cylindrical 2D crystals of this protein by treating outer membranes of N. crassa mitochondria with phospholipase A2. Using low-dose electron microscopy and image processing, we have generated a low-resolution (1.7nm) 3D structure of the channel in aurothioglucose. We want to extend this structure to higher resolution by using electron diffraction on frozen-hydrated specimens to collect Fourier amplitude information, which would be phased by low-resolution images and phase extension procedures being worked out in collaboration with Doug Dorset (Hauptmann-Woodward Foundation, Buffalo, NY). The cylindrical crystals of VDAC are small, and reflections from the two membrane layers often overlap, which limits the usefulness of these crystals for electron diffraction (ED) experiments. However, we have found ways to open these vesicles into flat single-layer sheets that approach 1 micrometer in linear dimension. We will determine the quality of the ED data from these sheets in frozen-hydrated state and assess whether they are suitable for a serious attempt at 3D data collection. The quality of ED data collected at 100, 400 and 1000 KV will be compared to determine whether direct-phasing approaches are feasible, i.e., whether expected improvements in Ewald sphere flattening and reduced dynamical scattering make a significant difference when attempting to apply direct phasing approaches to protein structure determination. Mitochondrial outer membranes are isolated, and VDAC crystals are prepared by removal of some of the membrane lipid using phospholipase A (PLA, from bee ven om). The membrane crystals are changed from their vesicular form into flat sheets by lowering the pH to 3.0 using either phosphate, citrate and glycine buffers. Fractions with high enzyme levels form crystals first, and crystals are harvested from fractions with lower enzyme levels on subsequent days. The crystals must be negatively stained or plunge-frozen on EM grids soon after they form, otherwise the residual enzyme will destroy them. Tests of the buffers, and of enzyme inhibitors to stabilize the crystals in the sheet form, are underway. Crystals in both vesicular and sheet forms are examined first with negative stain on a CTEM and the HVEM, then using cryo-EM on the CTEM. When results look promising, comparisons will be made using cryo-EM on the HVEM and IVEM. The diffraction and imaging conditions for the crystals were worked out for both the CTEM and the HVEM, and diffraction spots were observed on negatively-stained crystals. The first cryo-EM work is now being done.