Clinton S. Potter - US grants

9. Administration and Management Abstract We describe the Administrative and Management aspects of NRAMM which are designed to ensure that the Resource functions as a well-integrated multidisciplinary team.

6. Collaborative and Service Projects Abstract We will provide access to the advanced technologies, facilities, and infrastructure created at NRAMM to the national research community. This will include access to instrumentation as well as methods and expertize. Projects encompassed under collaborative and service include providing technical assistance to other laboratories in implementing NRAMM software and providing on-site access to instrumentation, staff and other resources. NRAMM's location at the Simons Electron Microscopy Center provides our Collaborative and Service Projects with access to a remarkable array of instruments and technology that are currently unavailable to many of our research users at their own institutions.

1. Overall: The National Resource for Automated Molecular Microscopy Abstract The overall mission of the National Resource for Automated Molecular Microscopy (NRAMM) is to develop, test and apply technology aimed at automating and streamlining cryo-electron microscopy (cryoEM) for structural biology. Our goal from the outset was to provide a pipeline for molecular microscopy that puts it on a par with other structural techniques like X-ray crystallography, so that, once suitable samples are in hand, getting to a high resolution 3D map is a straightforward and rapid undertaking. This one time dream is now obtainable for well-behaved samples as a result of many innovations and improvements in hardware and software, including most recently the advent of a new generation of cameras that directly detect electrons and record high frame rate movies allowing for correction of sample movement during image exposure. There is still work to be done however in ensuring that this method can be applied to the most challenging and biologically interesting samples and serve a very large influx of new practitioners of this method. Our Technology Research and Development (TRD) Projects for the next five years are designed to meet the challenges. Our goals are to develop novel approaches and promote their widespread use, as well as to integrate complex technologies into an efficient and effective method. To achieve these goals, we will focus on three Technological Research and Development Projects that encompass both completely new approaches as well as dynamic evolution of our current technologies. In TRD#1, we will seek to address the critical need to improve and automate vitrified sample preparation; develop methods for time resolved vitrification; and greatly accelerate the throughput of negatively stained sample screening. In TRD#2, we will continue to optimize the performance of both high and mid-range instruments, particularly with regard to integrating and assessing the value of several major accessories; we will develop a high-throughput data acquisition pipeline to support negative stain screening; and we will continue to develop enabling tools for data assessment, processing, analysis and reconstruction. Finally, in TRD#3 we will embark on the development of a new automated and streamlined pipeline for support of in-situ molecular microscopy, that is visualizing molecular structures inside cells. The technological themes at the heart of this proposal support our mission of providing an automated and streamlined pipeline for molecular microscopy that provides data of the highest possible quality and promotes the accessibility of the method to the wider scientific community. This mission is driven by close interactions and feedback from Driving Biological projects (DBPs), and further tested and validated by Collaborative and Service Projects (CSPs). We will also continue to work to maintain excellence in the areas of training and dissemination to promote the broadest possible use of these technologies.

Summary We propose to establish the National Center for CryoEM Access and Training (NCCAT) to be housed at the Simons Electron Microscopy Center (SEMC), which is a part of the New York Structural Biology Center (NYSBC), based in New York, NY. NCCAT will provide access to state-of-the-art equipment, including specimen preparation robots, high-end microscopes and direct detectors, screening microscopes, and all the other ancillary equipment required to solve structures to the highest possible resolution using cryo electron microscopy (cryoEM) methods. A highly qualified technical team will provide direct support, guidance and assistance to ensure that structures of the highest quality are produced in the shortest possible time. An already well established cross-training program will be expanded and extended to provide training across a wide variety of skill levels and career goals. NCCAT will adopt an open and transparent application process to ensure equal-opportunity nationwide access. The operations at NCCAT will build on already established practices at SEMC/NYSBC and benefit from the overall vibrant environment as well as the expertise of many of the highly trained staff already in place. NCCAT will be housed in new, custom-built space that will provide optimal, environmentally stable conditions. The major instrumentation will consist of 3 Titan Krios TEMs equipped with direct detectors, energy filters and phase plates, and 3 screening microscopes equipped with CMOS cameras. Streamlined workflows, already well established, will expanded to ensure that we collect the highest quality data as efficiently as possible. A user service operation will provide on-site, remote-access, and mail-in data collection services with fair and equal access to a nationwide user base. We will guide and assist users with all aspects of cryoEM, including sample preparation, data collection and image processing. A training program will encompass a full range of expectations, from the basics required to ensure that users produce valid structures, through more detailed training for users seeking independence, to fully embedded researchers who will be expected to graduate to running their own cryoEM research laboratories. While we anticipate that we will initially be called to serve and train mostly in the area of single particle cryoEM, we are well positioned to provide support in the area of cryoET that we predict will grow rapidly. While the goals of NCCAT are not innately innovative it is our intention to set the standard as one of the first world class, nationally accessible cryoEM access and training facilities that is freely and fairly available to all researchers in the US, and to continually strive to enable science of the highest quality and value.

7. Training Abstract The technologies, methods, and software developed at NRAMM will be transferred to both specialists and non- specialists though a series of activities including amongst others, on-site and small group training, topic focused workshops and a large biennial international workshop.

2. TRD#1: New Approaches to Specimen Preparation Abstract We are developing novel methods for specimen preparation that provide several advantages over traditional methods. These advantages include consumption of very small amounts of sample, and providing for more control over the quality of the prepared grids. The system uses a robotic device, called Spotiton, to dispense drops with volumes on the order of nanoliters to precise locations on an EM grid. The success of the device is coupled to a new ?self-blotting? grid that we have developed to provide a method for spreading sample to a thin film without the use of externally applied filter paper. Over the next 5 years we will design, develop, test and validate a generalized Spotiton system capable of multiple tasks including making well controlled vitrified grids, managing time resolved and fast mixing experiments, and preparing ~100 independent negatively stained samples on a single grid. We anticipate that a version of this device will be manufactured by a commercial company so that it becomes generally available to the research community.

Summary We propose to establish the National Center for In-situ Tomographic Ultramicroscopy (NCITU) to be based at the Simons Electron Microscopy Center (SEMC), which is a part of the New York Structural Biology Center (NYSBC), based in New York, NY. NCITU will be dedicated to developing and providing access to both standard and advanced methods for in-situ cryoET specimen preparation, and to disseminating this expertise through a cryoET cross-training program designed to serve a wide variety of skill levels and career goals. The primary goal of NCITU will be to provide user access and service towards preparing specimen for direct visualization of macromolecular associations and organization, particularly in native cells. A highly qualified technical team will provide direct hands-on service, guidance, and assistance to ensure that samples and grids are prepared with the highest quality and in the shortest time possible. An already well established cross-training program will be expanded and extended to provide training in every aspect of the cryoET specimen preparation pipeline. The operations at NCITU will build on already established practices at SEMC/NYSBC and benefit from the overall vibrant environment as well as the expertise of the highly trained staff already in place. NCITU will provide a full range of services for in-situ sample preparation for tomography including: culturing biological samples, plunge freezing and high pressure freezing, cryo-light microscopy (cryoLM), cryo- ultramicrotomy, focused ion beam milling (FIB-milling) including lift-out, and initial cryoTEM screening prior to submission of specimens to the network Hub for high-resolution cryoET data collection. We have already established workflows for many of these activities and have active research projects focused on expanding capabilities for automating and streamlining the entire workflow. We will continue to stay at the forefront of new developments in this area and we will incorporate new methods, technologies and workflows into the standard NCITU operating procedures as soon as these are stable and robust. While the goals of NCITU are not innately innovative, it is our intention to set the standard as one of the first world class, nationally accessible cryoET sample preparation facilities, to provide access and training that is freely and fairly available to all researchers in the US, and to continually strive to improve methods and enable science of the highest quality and value.