As atomic structures of membrane proteins are readily resolved and predicted due to the recent advances in biophysical methods like cryo-EM as well as computational tools like AlphaFold, there emerges an urgent need to investigate the real-time structural dynamics at the single molecule level to obtain novel insights in the membrane structural biology. Here, we present a unique sample preparation method for high-speed atomic force microscopy (HS-AFM), which enabled us to immobilize membrane proteins in an extended lipid bilayer, while preserving their dynamics. This strategy bypasses the previous requirement of the dense packing of molecules. The approach allowed to acquire HS-AFM movies of individual transporters with increased resolution and in a physiologically more relevant setting. Using GltPh, a trimeric aspartate transporter, as a model protein, we demonstrate that HS-AFM combined with the presented strategy was able to track structural dynamics of individual protomers at sub-second temporal resolution and sub-nanometer spatial resolution for tens of seconds. Recent cryo-EM studies of GltPh revealed two major states of the protein, outward-facing (OF) and inward-facing (IF) states, as well as several conformational sub-states. Real-time transitions between these states were observed in the HS-AFM data with unprecedented detail. For data analysis, we developed a principal component analysis (PCA) based method to sort protomers at each time point into different conformations according to their structural features, reconstructing a structure-time trace for each protomer of a single molecule. Using the previously developed localization AFM (LAFM) method, we calculate Angstrom-resolution GltPh surface structures of single molecules as it transits through several conformational states in the transport cycle. The presented method enables direct observation and analysis of the structure and dynamics of individual unlabeled membrane proteins, unlocking the potential of HS-AFM in the study of single molecule structural biology.