MACCHESS PROGRAM FOR PRESSURE CRYOCOOLING AND RELATED PROCEDURES

压力低温冷却的 MACCHESS 程序及相关程序

• 批准号: 8171530
• 负责人: HARRY F NOLLER
• 金额: $ 1.37万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171530
• 关键词: Address; Apoptosis; Area; Automation; Beryllium; Biological; Bite; Buffaloes; Cells; Collaborations; Communities; Complex; Computer Retrieval of Information on Scientific Projects Database; Data Collection; Dehydration; Dependence; Development; Diamond; Drops; Environment; Equipment Safety; Explosion; Figs - dietary; Funding; Future; Gases; Goals; Grant; Helium; High temperature of physical object; Housing; Institutes; Institution; Lead; Length; Liquid substance; Literature; Magnetism; Methodology; Methods; Mission; Modification; Nitrogen; Noble Gases; Oils; Phase; Physics; Physiological; Plumbing; Pressure Transducers; Procedures; Process; Protein Dynamics; Protein Structure Initiative; Proteins; Protocols documentation; Publications; Recording of previous events; Reproduction; Research; Research Personnel; Resources; Ribosomes; Safety; Screening procedure; Signaling Protein; Site; Source; Steel; Techniques; Temperature; Testing; Thermodynamics; Time; Training; United States National Institutes of Health; Work; base; cryogenics; falls; hazard; improved; innovation; interest; medical schools; member; novel; novel strategies; operation; pressure; prevent; programs; protein structure; prototype; research study; structural biology; success; three dimensional structure; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The primary focus of this initiative is on developing new approaches for preparing crystals to yield high-quality diffraction, thus enabling detailed structural analysis of challenging and extremely important questions of fundamental biological interest. Collaborative experiments will address two such biological problems in particular: (1) structural studies of a functional ribosome unit, in collaboration with Dr. Harry Noller (U.C. Santa Cruz) and (2) structural analysis of signaling proteins responsible for the programmed death of cells (apoptosis), in collaboration with Dr. Hao Wu (Cornell-Weill Medical College). In addition, we will investigate the use of pressure as a thermodynamic parameter to study the mechanisms of protein dynamics, function, and interaction, as well as seeking a fundamental understanding of how proteins assume and maintain their three dimensional structures; Dr. Sol Gruner will lead this effort. Three basic methodologies will be used: (1) Cryocooling crystals under high pressure for ambient pressure diffraction at cryogenic temperatures, (2) diffraction of pressurized crystals at physiological temperatures and high pressures using beryllium and diamond pressure vessels, and (3) application of novel flash-cooling and annealing techniques, using protocols developed by a Cornell technical collaborator, Dr. Rob Thorne. Cornell and MacCHESS have a long history of innovations in the field of crystal cryocooling, beginning with the development of cryoloops by T-Y Teng in 1985 [5]. Currently, groups led by Sol Gruner and Rob Thorne are actively investigating cryocooling techniques, with support from MacCHESS. Over the next five years, we, together with our collaborators, propose to focus on this area to develop apparatus and techniques for handling crystals under various conditions, particularly at high pressures. Our overall goals will be (1) to permit MacCHESS users to obtain the best possible diffraction from their crystals, (2) to increase the success rate for noble-gas phasing, and (3) to further investigate the dependence of protein structure on pressure and temperature. Dissemination of the new methodologies, and training of the larger MacCHESS user community, is occurring now, and will continue to occur concurrently with future developments. Pressure cryocooling apparatus development The pressure-cryocooling apparatus developed so far is a prototype that has worked well for initial experiments [18]. Briefly, crystals are picked up in a cryoloop with an attached magnetic steel wire in a droplet of Hampton NVH oil to prevent dehydration. These are loaded into the high pressure cryocooling apparatus consisting of commercial high pressure plumbing and pressure transducers. The apparatus containing the crystal is then pressurized with helium gas to pressures in the 100 - 200 MPa range. Once at high pressure, a magnetic constraint is released and the crystals fall down a length of high pressure tubing into a cold zone kept at LN2 temperature. The helium pressure is released and the crystals are thereafter handled at ambient pressure, in the same manner as normal flash-cryocooled crystals for cryocrystallographic data collection. The crystals are indefinitely stable as long as they are not allowed to warm above ~130¿ K. The process for Kr or Xe phasing is a bit more complex. First, crystals are loaded into the apparatus as in the previous paragraph. The crystals are then pressurized with Kr or Xe gas to 10 MPa. After an equilibration time, the compressed gas is released and the crystals are re-pressurized with helium. The apparatus is then pressurized to 100  200 MPa over the course of a few minutes, after which the magnetic constraint is released and crystals are dropped into a cold zone at liquid nitrogen temperature. With the publication of the pressure-cryocooling method [18], many crystallographers have expressed interest in trying it on their crystals, and as of December 2006 about two dozen groups have collaborated to do so. Results have been encouraging and are beginning to appear in the literature [12]. However, it has become clear that the existing apparatus needs to be improved for efficiency and safety and to extend experimental capabilities. Gas at several hundred MPa presents significant explosion hazards and must be handled very carefully. The prototype apparatus is housed in Dr. Gruner's lab in Cornell's Physics Department, and is not suitable for a general user facility. There have also been requests to reproduce the apparatus at the NSLS and the APS. Accordingly, as part of the dissemination mission of MacCHESS, we are including ease of reproduction as a goal in future redesigns of the apparatus. A redesigned cryocooling apparatus is now being commissioned (Fig. 30). Improvements include: (1) Pressure capability to 400 MPa. The old apparatus was limited to 200 MPa. (2) A more robust, larger, and easier to use safety enclosure to enable various cryocooling protocols. (3) Automation to reduce the time between Kr or Xe gas exposure and pressure cryocooling. (4) Modifications to be able to process more crystals at a time. The redesigned apparatus will be tested in Dr. Gruner's physics department lab, which will also be the site for experimentation to extend the technique and develop robust usage protocols (see below). We propose to build a second apparatus to be installed at CHESS for MacCHESS users using these protocols. The MacCHESS technical operations staff will be trained to operate this machinery. A third apparatus will likely soon be installed at the NSLS, and others may follow at other institutions. New protocols developed in Dr. Gruner's lab will be disseminated to other installations as part of the Mac- CHESS dissemination mission. High pressure gas involves significant safety issues, but we are confident that these can be dealt with in the CHESS user environment. Procedures have already been developed at CHESS for using a high pressure gas-loading apparatus for diamond anvil cells; the new apparatus will use some of the same gas-handling equipment and safety precautions. Protocols will need to be developed for inexperienced users. The pressure cryocooling apparatus at CHESS will be a research tool, used largely for crystallographically difficult proteins, i.e., those where other methods have not worked. Note that this effort is distinct from, yet synergistic with, an effort at CHESS (as a member of the High Throughput Center for Structural Biology, a Protein Structure Initiative center based at the Hauptmann-Woodward Institute in Buffalo, NY) to develop a high-throughput pressure cryocooling apparatus for crystal screening.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 该计划的主要重点是开发准备晶体以产生高质量衍射的新方法，从而实现了挑战的详细结构分析以及基本生物学兴趣的极为重要的问题。协作实验将特别解决两个这样的生物学问题：（1）与Harry Noller博士（U.C. Santa Cruz）合作的功能性核糖体单元的结构研究，以及（2）与Hao Wu Wu（Cornell Weell Weill Medical Collecton）合作，负责细胞（凋亡）的信号蛋白的结构分析。此外，我们将研究压力用作热力学参数，以研究蛋白质动力学，功能和相互作用的机制，并寻求对蛋白质如何假设和维持其三维结构的基本了解； Sol Gruner博士将领导这一努力。将使用三种基本方法：（1）在高压下为低温温度下的环境压力衍射的冷冻冷却晶体，（2）在物理温度下加压晶体的衍射和使用贝莱尔和钻石压力容器的高压，以及（3）使用新颖的闪光冷却技术和使用方案，使用康奈尔技术合作的协议来实现新颖的闪存和退火技术。康奈尔（Cornell）和麦克切斯（MacChess）在Crystal Cryocooling领域具有悠久的创新历史，从1985年T-Y Teng的冷冻冷却开始[5]。目前，在MacChess的支持下，由Sol Gruner和Rob Thorne领导的小组正在积极调查冷冻技术。在接下来的五年中，我们与我们的合作者一起，提议专注于这一领域，以开发在各种条件下，尤其是在高压下处理晶体的设备和技术。我们的总体目标是（1）允许MacChess用户从其晶体中获得最佳的衍射，（2）提高贵族 - 气体相位的成功率，以及（3）进一步研究蛋白质结构对压力和温度的依赖性。现在正在发生新方法的传播以及较大的Macchess用户社区的培训，并将继续与未来的发展同时发生。到目前为止，压力冷冻设备开发到迄今为止开发的压力 - 晶型设备是一个原型，它在初始实验中效果很好[18]。简而言之，将晶体与冰冻的冻结液中捡起，并在汉普顿NVH油的一滴液滴中捡起晶体，以防止脱水。这些被加载到由商业高压管道和压力传感器组成的高压冷冻冷却设备中。然后将包含晶体的设备用氦气加压到100-200 MPa范围内的压力。一旦在高压下，就会释放出磁性约束，晶体降低了一定长度的高压管进入冷区，将其保持在LN2温度下。氦气压力被释放，然后在环境压力下以与正常的闪存晶体晶体相同的方式处理晶体，以进行冷冻晶体学数据收集。只要不允许晶体在〜130¿K上方加热，这些晶体就可以无限期地稳定。KR或XE相位的过程更为复杂。首先，像上一段一样将晶体加载到设备中。然后将晶体用KR或XE气体加压至10 MPa。平衡时间后，释放压缩气体，并用氦气重新压榨晶体。然后在几分钟的时间内将设备加压至100 200 MPa，然后在液态氮温度下释放磁性约束并将晶体掉入冷区。通过出版了压力 - 晶方法[18]，许多晶体学者对尝试晶体表示兴趣，截至2006年12月，大约有两个十二个群体合作了。结果令人鼓舞，并开始出现在文献中[12]。但是，很明显，为了提高效率和安全性并扩展实验能力，现有设备需要改进。数百MPA的气体呈现出重大的爆炸危害，必须非常谨慎地处理。该原型设备位于康奈尔（Cornell）物理系的Gruner博士实验室中，不适合通用用户设施。还要求在NSL和APS上复制设备。根据Macchess的传播使命的一部分，我们将易于繁殖作为设备的未来重新设计的目标。重新设计的冷冻机器现在正在委托（图30）。改进包括：（1）压力能力为400 MPa。旧设备仅限于200 MPa。 （2）更强大，更大且易于使用安全外壳以实现各种冷冻协议。 （3）自动化以减少KR或XE气体暴露与压力冷冻冷却的时间。 （4）修改一次可以处理更多的晶体。重新设计的设备将在Gruner博士的物理部门实验室中进行测试，这也将是扩展技术并开发强大用法协议的实验的站点（请参阅下文）。我们建议使用这些协议为MacChess用户构建第二个设备，以安装国际象棋。 MacChess技术运营人员将接受培训以操作此机械。 NSL可能很快就会安装第三个设备，其他机构可能会在其他机构遵循。作为麦克索斯传播任务的一部分，在格鲁纳博士实验室中开发的新协议将被传播到其他设施中。高压气体涉及重大安全问题，但我们相信可以在国际象棋用户环境中处理这些问题。在国际象棋中已经开发了用于使用高压气体载体钻石细胞的机构。新设备将使用一些相同的气体处理设备和安全预防措施。需要为没有经验的用户开发协议。国际象棋的压力冷冻冷却设备将是一种研究工具，主要用于晶体学上困难的蛋白质，即这些其他方法尚未起作用。请注意，这项工作与纽约州布法罗Hauptmann-Woodward Institute的蛋白质结构生物学中心（作为High吞吐量结构生物学中心的成员）的努力不同，但与之协同作用。