Development and Deployment of New Structure Prediction and Determination Capabilities at the UCLA-DOE Institute
Authors:
Jose A. Rodriguez* ([email protected]), Samantha Zink, Jeff Qu, Niko Vlahakis, Theodosia Bartashevitch, Lukasz Salwinski, Roger Castells-Graells, Todd O. Yeates, David Eisenberg, Matteo Pellegrini (PI)
Institutions:
UCLA-DOE Institute for Genomics and Proteomics, University of California–Los Angeles
URLs:
Goals
Research in the DOE-University of California–Los Angeles (UCLA) Institute for Genomics and Proteomics (IGP) includes major efforts in the area of imaging science, proteomics, structure prediction and atomic structure determination. These new capabilities help scientists better understand microbial biosystems, their genomics and molecular biology. This team is pioneering new enabling capabilities that facilitate the discovery of molecular structural features affecting protein function and specificity, to better understanding of bioenergy crops and microbes. These capabilities span the broad areas of X-ray diffraction, electron microscopy, and micro-electron diffraction (MicroED), along with computational structure and function prediction methods. This team is also enabling rapid access to robust public-facing tools for use by the BER community. This group’s efforts in imaging science and protein characterization bridge a number of technological areas to address pressing problems in protein structure and function.
Abstract
Breakthroughs in cryo-electron microscopy (cryo-EM): Numerous technical advances have made cryo-EM an attractive method for atomic structure determination. Cryo-EM is ideally suited for very large structures; symmetrical structures like viruses are especially amenable. However, problems of low-signalto-noise in imaging small proteins makes it practically impossible to determine structures smaller than about 50 kilodaltons, leaving a great many cellular proteins and enzymes (and nucleic acid molecules) outside the reach of this important structural technique. The DOE-UCLA IGP team has broken through this barrier by engineering novel scaffolds with sufficient rigidity and modularity to achieve resolution useful for interpreting atomic structure. This team has applied this system to image a 19 kDa protein, obtaining multiple structures of its sequence variants unbound and bound to a small molecule. The findings highlight the promise of these novel scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases as well as other important targets. Recent efforts have been aimed at imaging microbial and plant protein targets.
Enabling microcrystal electron diffraction (MicroED) methods: A broad array of atomic structures has now been determined by MicroED; they include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. This team is further enhancing the capabilities of electron diffraction (ED) by improving understanding of electron counting detectors and their application to diffraction measurements. In addition, the group is broadening comprehension of electron beam-induced radiation damage and its consequences for molecular systems and their characterization at atomic resolution. Collectively, these efforts have yielded new insights into how ED data are impacted by electron beam-induced lattice reorientation and the impact of radiation damage on the ability to determine the chiral nature of handed molecules.
Tools for analysis of condensate or aggregate-forming proteins: The recent revolution in artificial intelligence (AI) and machine learning methods has dramatically improved scientists’ ability to predict protein structure and sequence characteristics. This team has exploited the growing capacity of AI models to train a fully connected neural network to emulate the predictive abilities of computationally time-consuming 3D profiling approaches. This method relies on the network to calculate the propensity of segments in a sequence to form amyloid-like contacts or structures. Whereas the previous approach required weeks or months of compute time to evaluate an entire proteome, the new approach can evaluate the entire yeast proteome in 15 minutes and is available as an online server for public use.
The institute’s enabling capabilities will broadly facilitate the determination and prediction of unknown macromolecular structures with importance for bioenergy.
References
This work is supported by DOE grant DE-FC02-02ER63421.
Funding Information
Castells-Graells, R., et al. 2023. “Cryo-EM Structure Determination of Small Therapeutic Protein Targets at 3 Å- Resolution Using a Rigid Imaging Scaffold,” Proceedings of the National Academy of Sciences of the United States of America 120(37), e2305494120. DOI:10.1073/ pnas.2305494120.
Thompson, M. C., et al. 2020. “Advances in Methods for Atomic Resolution Macromolecular Structure Determination,” F1000Research 9. DOI:10.12688/ f1000research.25097.1.