This study aims to enhance the understanding of G-quadruplex (G4) structures by analyzing 333 intramolecular G4s using the ASC-G4 software. The analysis reveals key concepts and new information, including: 1. **Topological Distinctions**: The study introduces eight distinguishable topologies, each with a unique groove-width signature and glycosidic configuration (gc) pattern. The relative orientations of the stacking guanines within the strands determine the twist and tilt of the helices, which impact the minimum groove widths. 2. **Glycosidic Configurations**: The gc progression along the strands is crucial for understanding the topology and groove widths. The vertical gc succession has no strict one-to-one relationship with the topology, leading to discrepancies in circular dichroism (CD) spectra. 3. **New Concept of Platypus G4s**: These are structures with properties corresponding to multiple topologies, which were not previously recognized. 4. **Grove Width Analysis**: The study quantifies the groove widths for narrow, medium, and wide grooves, providing specific values for each type. The groove widths are influenced by the direction of the strands and the gc patterns. 5. **Ligand and Protein Effects**: The presence of ligands and proteins does not significantly affect the groove widths if the topology remains unchanged. However, the presence of a ligand can modify the G4 structure, leading to different topologies. 6. **GC Pattern Correlation**: In regular G4s, the gc pattern of adjacent guanines within a tetrad is correlated with the strand directions. This correlation explains the observed groove widths and gc patterns. The study provides a comprehensive analysis of G4 structures, highlighting the importance of topological distinctions and the impact of strand directions on groove widths and gc patterns.This study aims to enhance the understanding of G-quadruplex (G4) structures by analyzing 333 intramolecular G4s using the ASC-G4 software. The analysis reveals key concepts and new information, including: 1. **Topological Distinctions**: The study introduces eight distinguishable topologies, each with a unique groove-width signature and glycosidic configuration (gc) pattern. The relative orientations of the stacking guanines within the strands determine the twist and tilt of the helices, which impact the minimum groove widths. 2. **Glycosidic Configurations**: The gc progression along the strands is crucial for understanding the topology and groove widths. The vertical gc succession has no strict one-to-one relationship with the topology, leading to discrepancies in circular dichroism (CD) spectra. 3. **New Concept of Platypus G4s**: These are structures with properties corresponding to multiple topologies, which were not previously recognized. 4. **Grove Width Analysis**: The study quantifies the groove widths for narrow, medium, and wide grooves, providing specific values for each type. The groove widths are influenced by the direction of the strands and the gc patterns. 5. **Ligand and Protein Effects**: The presence of ligands and proteins does not significantly affect the groove widths if the topology remains unchanged. However, the presence of a ligand can modify the G4 structure, leading to different topologies. 6. **GC Pattern Correlation**: In regular G4s, the gc pattern of adjacent guanines within a tetrad is correlated with the strand directions. This correlation explains the observed groove widths and gc patterns. The study provides a comprehensive analysis of G4 structures, highlighting the importance of topological distinctions and the impact of strand directions on groove widths and gc patterns.