Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes.
The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.
Yin
Chang-Cheng
CC
Department of Biophysics, Peking University Health Science Center, Peking University, 38 Xueyuan Road, Beijing 100083, PR China. ccyin@bjmu.edu.cn
Han
Hongmei
H
Wei
Risheng
R
Lai
F Anthony
FA
D000595:Amino Acid Sequence; D000818:Animals; D003460:Crystallization; D005583:Fourier Analysis; D008055:Lipids / Q000737:chemistry; D008566:Membranes / Q000737:chemistry / Q000648:ultrastructure; D008956:Models, Chemical; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure
D008055:Lipids; D019837:Ryanodine Receptor Calcium Release Channel