The successful development of an automated cryoEM pipeline for helical specimens in the previous grant period and the subsequent establishment of the National Resource for Automated Molecular Microscopy sets the stage for this current application. One of the critical missing components in automated cryoEM is the means to determine unambiguous initial models for an unknown macromolecular complex and the infrastructure to analyze the degree of conformational heterogeneity in the specimen. The ability to determine the structure of macromolecular complexes in a variety of conformational states is the next big challenge for cryoEM, representing a unique opportunity to address interesting biological questions. Our contribution to this challenge will be to design, implement and test a system for single particle structural analysis that uses experimental, rather than computational, strategies for calculating an initial model, determining the degree of conformational and orientational variability in a specimen, and rejecting imperfect particles during refinement. To achieve this we will develop procedures to determine sets of initial models using both tomographic and random conical data acquisition methods, and procedures for large scale data collection of tilted pairs of images. The data acquisition and analysis procedures will be supported by a web based graphical user interface that will be tightly integrated with a relational database. This work represents a significant extension of the current efforts underway at the National Resource for Automated Molecular Microscopy (NRAMM).
| Cheng, Anchi (2013) Automated grid handling and image acquisition for two-dimensional crystal screening. Methods Mol Biol 955:297-305 |
| Cheng, Anchi (2013) Automation of data acquisition in electron crystallography. Methods Mol Biol 955:307-12 |
| Lander, Gabriel C; Baudoux, Anne-Claire; Azam, Farooq et al. (2012) Capsomer dynamics and stabilization in the T = 12 marine bacteriophage SIO-2 and its procapsid studied by CryoEM. Structure 20:498-503 |
| Gibbons, Brian J; Brignole, Edward J; Azubel, Maia et al. (2012) Subunit architecture of general transcription factor TFIIH. Proc Natl Acad Sci U S A 109:1949-54 |
| Lyumkis, Dmitry; Moeller, Arne; Cheng, Anchi et al. (2010) Automation in single-particle electron microscopy connecting the pieces. Methods Enzymol 483:291-338 |
| Yoshioka, Craig; Carragher, Bridget; Potter, Clinton S (2010) Cryomesh: a new substrate for cryo-electron microscopy. Microsc Microanal 16:43-53 |
| Mulder, Anke M; Yoshioka, Craig; Beck, Andrea H et al. (2010) Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit. Science 330:673-7 |
| Voss, Neil R; Lyumkis, Dmitry; Cheng, Anchi et al. (2010) A toolbox for ab initio 3-D reconstructions in single-particle electron microscopy. J Struct Biol 169:389-98 |
| Lander, Gabriel C; Stagg, Scott M; Voss, Neil R et al. (2009) Appion: an integrated, database-driven pipeline to facilitate EM image processing. J Struct Biol 166:95-102 |
| Suloway, Christian; Shi, Jian; Cheng, Anchi et al. (2009) Fully automated, sequential tilt-series acquisition with Leginon. J Struct Biol 167:11-8 |
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