Project Summary We propose to explore and document structural heterogeneities in virus capsid populations, using a combination of computer simulations and the re-examination of raw cryo-electron microscopy (cryoEM) data. Although structural biology methods by design typically yield virus capsid models that are free of defects, both in vivo and in vitro capsid populations exhibit structural heterogeneities of probable biological and potential therapeutic importance. Characterizing and understanding meta-stable structures that are partially or mis- assembled could lead to strategies for retarding assembly by inducing these off-pathway states. We propose to focus on hepatitis B virus (HBV) as a model system for this exploratory R21. HBV has previously been studied by the PIs using both computational and experimental approaches. Methodologically, we will computationally generate a large set of three-dimensional models of defective HBV capsids which will be filtered in an iterative fashion by comparison to existing two-dimensional cryoEM images. The models will be generated both by (i) assembly and disassembly simulations and (ii) combinatorial removals of capsomers from the fully symmetric structure. Simulations will employ the recently developed tabulation approach which has enabled assembly simulations at an unprecedented level of structural detail; additionally, the weighted ensemble enhanced sampling approach will be employed, as it has been shown to yield detailed assembly pathways. Structural models which are validated by comparison to cryoEM data will be made publicly accessible. The basic idea of the proposal may be seen as mirroring the change in perspective which has taken hold in single-molecule structural biology: it is now widely accepted that understanding the function of complex biomolecules requires an `ensemble picture' of multiple structures representing functional pathways and motions. We believe that heterogeneity in virus capsid structural behavior could be equally important and merits careful investigation. This proposal is an attempt to initiate an in-depth investigation into capsid defects, which could be of long-term therapeutic importance.