Handling complexity in huge heterogeneous cryo electron microscopy datasets: visualising biological processes at quasi-atomic resolution

溶液中的生物分子是生命的基础。生物大分子在水环境中相互作用，形成具有不同构象状态的络合物来执行其生物功能。将生物溶液快速冻结到液氮温度，使溶液凝固成玻璃状的水(玻璃水)，从而完美地保存生物复合体。生物络合物的玻璃化溶液可以直接在电子显微镜的低温样品架上成像。然后，从获得的图像计算嵌入复合体的三维(3D)结构。整个过程被称为“冷冻-EM”。然而，尽管在玻璃化溶液中展示了出色的样品，但低温EM产生的原始图像噪音极大，难以处理。这是因为生物样品对辐射高度敏感，在接受10el/ä2的电子曝光后就开始衰变。在如此低的曝光水平下，图像中的量子噪声超过了分子信息的数量级。申请者小组一直是开发3D低温电磁方法的领导小组之一。CRYO-EM最近在分辨率水平上取得了令人印象深刻的结果，分辨率高于~10？，一些高度对称的病毒衣壳结构被揭示到~4？分辨率。因此，单粒子技术现在正接近以前只能通过X射线结晶学才能达到的分辨率水平。对结构进行可靠的从头原子解释是可能的，到目前为止，分辨率主要是通过X射线结晶学实现的，其中生物分子必须限于晶体的刚性。相比之下，单粒子冷冻-EM方法提供了一个直接窗口，可以了解溶液中发生的事情的全部生物复杂性。低温电子显微镜图像可能包含过多的处于不同功能状态的不同络合物。在现代生物学中，以准原子分辨率从极其嘈杂的数据中提取结构信息是一个挑战。CRYO-EM有可能提供分辨率为~3？的信息。为了实现在行动中观察生物机械的这一新水平的分辨率，我们首先需要优秀的最先进的电磁仪器和有效的数据收集管道来获取所需的巨大数据集。最重要的是，我们需要创造快速、大规模并行的新的和改进的计算工具。我们新的像散数据收集策略显著增加了单粒子图像的高分辨率信息含量，并且已经导致了~4？分辨率结构。散光数据收集也将使我们能够处理小的~100kD蛋白质，这个大小到目前为止还不适合单粒子处理。我们的新的并行特征向量-特征值‘MSA’方法能够处理TB大小的数据集，将在各种新的程序中使用，包括我们新的‘总数据集’传递函数(CTF)校正。新的并行MSA模式识别程序也将被开发，用于从玻璃化溶液中的噪声图像中分离不同构象状态的不同络合物。通过将新的整体程序并行化并将其映射到快速的新计算硬件上，我们希望极大地提高可用的绝对计算能力。方法开发人员的专家团队已经就位。先进的电磁仪器集中在CBEM国家冷冻-EM设施中，该设施容纳了CM 300液氦仪器。为了测试我们在最新低温电磁技术上的程序，我们可以使用世界上首批可用的FEI Titan Krios仪器之一。通过低温电子显微镜对复杂的生物机械活动的直接可视化可能会在我们对生物学的理解中开启新的篇章。

Biological molecules in solution are the basis of life. Biological macromolecules interact with each other in an aqueous environment, forming complexes that have different conformational states in performing their biological function. Rapid freezing of biological solutions to liquid nitrogen temperatures solidifies the solution to a glassy phase of water ('vitrified' water) that perfectly preserves biological complexes. Vitrified solutions of biological complexes can be imaged directly in the cryogenic specimen holder of the electron microscope. From the images obtained one then calculates three-dimensional (3D) structures of the embedded complexes. The whole procedure is known as 'cryo-EM'. However, in spite of the excellent specimen presentation inside the vitrified solution the raw images produced by cryo-EM are extremely noisy and difficult to process. This is because the biological samples are highly radiation sensitive and start decaying after receiving an electron exposure of only 10el/Å2. At such low exposure levels the 'quantum noise' in the images exceeds the molecular information by orders of magnitude. The group of the applicant has been one of the leading groups in developing the 3D cryo-EM methodology. Cryo-EM has recently yielded impressive results at resolution levels better than ~10Å, with some highly symmetrical viral capsids structures revealed up to ~4Å resolution. Single-particle techniques are thus now approaching resolution levels previously only achievable by X-ray crystallography. A reliable de-novo atomic interpretation of a structure is possible at ~3Å, resolutions hitherto achieved mainly by X-ray crystallography, where the biological molecules are necessarily confined to the rigidity of a crystal. Single-particle cryo-EM methods, in contrast, give a direct window into the full biological complexity of what is happening in solution. Cryo-EM images can contain a plethora of different complexes, in different functional states. Extracting that structural information from the extremely noisy data, at quasi-atomic resolution is a challenge in modern biology. Cryo-EM has the potential to provide information at resolution levels of ~3Å. To achieve this new level of resolution for observing biological machinery in action, we first need excellent state-of-the-art EM instrumentation and effective data-collection pipelines to harvest the required huge data sets. Above all we need to create fast, massively-parallel new and improved computational tools. Our new astigmatic data-collection strategy significantly increases the high-resolution information content of single-particle images and has already lead to a ~4Å resolution structure. Astigmatic data collection will also allow us to process small ~100kD proteins, a size hitherto not amenable to single-particle processing. Our new parallel eigenvector-eigenvalue 'MSA' methodology, capable of handling terabyte-sized data sets, will be exploited in various new procedures including our new 'total data set' transfer-function (CTF) correction. The new parallel MSA pattern recognition programs will also be exploited for separating the different complexes in their different conformational states from their noisy images in the vitrified solution. By the parallelisation of the new overall procedures and by mapping them onto fast new computing hardware, we want to greatly improve the sheer computing power available. The specialist team of method developers are already in place. Advanced EM instrumentation is in place centred in the CBEM national cryo-EM facility which houses the CM 300 liquid Helium instrument. For testing our procedures on the latest cryo-EM technology we have access to one of the first FEI Titan Krios instruments available world-wide. The direct visualization, by cryo-EM, of complex biological machinery in action may open a new chapter in our understanding of biology.