High-resolution Doppler radar velocities and in situ surface observations collected in a tornadic supercell on 5 June 2009 during the second Verification of the Origins of Rotation in Tornadoes Experiment (VORTEX2) are assimilated into a simulated convective storm using an ensemble Kalman filter (EnKF). A series of EnKF experiments using a 1-km horizontal model grid spacing demonstrates the sensitivity of the cold pool and kinematic structure of the storm to the assimilation of these observations and to different model microphysics parameterizations. An experiment is performed using a finer grid spacing (500 m) and the most optimal data assimilation and model configurations from the sensitivity tests to produce a realistically evolving storm. Analyses from this experiment are verified against dual-Doppler and in situ observations and are evaluated for their potential to confidently evaluate mesocyclone-scale processes in the storm using trajectory analysis and calculations of Lagrangian vorticity budgets. In Part II of this study, these analyses will be further evaluated to learn the roles that mesocyclone-scale processes play in tornado formation, maintenance, and decay. The coldness of the simulated low-level outflow is generally insensitive to the choice of certain microphysical parameterizations, likely owing to the vast quantity of kinematic and in situ thermodynamic observations assimilated. The three-dimensional EnKF wind fields and parcel trajectories resemble those retrieved from dual-Doppler observations within the storm, suggesting that realistic four-dimensional mesocyclone-scale processes are captured. However, potential errors are found in trajectories and Lagrangian three-dimensional vorticity budget calculations performed within the mesocyclone that may be due to the coarse (2 min) temporal resolution of the analyses. Therefore, caution must be exercised when interpreting trajectories in this area of the storm.