The CHARMM-GUI Membrane Builder is a web-based tool that allows users to build all-atom protein/membrane or membrane-only systems for molecular dynamics simulations through an automated process. This paper describes new features and major improvements in Membrane Builder that enable users to build more realistic biological membrane systems. These include the addition of over 180 lipid types, such as phosphoinositides, cardiolipin, sphingolipids, bacterial lipids, and ergosterol; an enhanced building procedure for lipid packing around proteins; a reliable algorithm to detect lipid tail penetration to ring structures and protein surfaces; a distance-based algorithm for faster ion displacement; CHARMM inputs for P21 image transformation; and NAMD equilibration and production inputs. The robustness of these features is demonstrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types. The simulation results show that the new features improve the accuracy and efficiency of building realistic biological membrane systems. The paper also discusses the application of Membrane Builder in simulating E. coli membranes and highlights the importance of lipid composition in membrane function. The future development of Membrane Builder will focus on incorporating more lipid types, such as glycolipids, glycophosphatidylinositol linkages, and lipopolysaccharides. The tool is expected to become a valuable resource for studying the structure and dynamics of proteins and lipids in biological membranes.The CHARMM-GUI Membrane Builder is a web-based tool that allows users to build all-atom protein/membrane or membrane-only systems for molecular dynamics simulations through an automated process. This paper describes new features and major improvements in Membrane Builder that enable users to build more realistic biological membrane systems. These include the addition of over 180 lipid types, such as phosphoinositides, cardiolipin, sphingolipids, bacterial lipids, and ergosterol; an enhanced building procedure for lipid packing around proteins; a reliable algorithm to detect lipid tail penetration to ring structures and protein surfaces; a distance-based algorithm for faster ion displacement; CHARMM inputs for P21 image transformation; and NAMD equilibration and production inputs. The robustness of these features is demonstrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types. The simulation results show that the new features improve the accuracy and efficiency of building realistic biological membrane systems. The paper also discusses the application of Membrane Builder in simulating E. coli membranes and highlights the importance of lipid composition in membrane function. The future development of Membrane Builder will focus on incorporating more lipid types, such as glycolipids, glycophosphatidylinositol linkages, and lipopolysaccharides. The tool is expected to become a valuable resource for studying the structure and dynamics of proteins and lipids in biological membranes.