De novo backbone trace of GroEL from single particle electron cryomicroscopy.
In this work, we employ single-particle electron cryo-microscopy (cryo-EM) to reconstruct GroEL to approximately 4 A resolution with both D7 and C7 symmetry. Using a newly developed skeletonization algorithm and secondary structure element identification in combination with sequence-based secondary structure prediction, we demonstrate that it is possible to achieve a de novo Calpha trace directly from a cryo-EM reconstruction. The topology of our backbone trace is completely accurate, though subtle alterations illustrate significant differences from existing crystal structures. In the map with C7 symmetry, the seven monomers in each ring are identical; however, the subunits have a subtly different structure in each ring, particularly in the equatorial domain. These differences include an asymmetric salt bridge, density in the nucleotide-binding pocket of only one ring, and small shifts in alpha helix positions. This asymmetric conformation is different from previous asymmetric structures, including GroES-bound GroEL, and may represent a "primed state" in the chaperonin pathway.
Ludtke
Steven J
SJ
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Baker
Matthew L
ML
Chen
Dong-Hua
DH
Song
Jiu-Li
JL
Chuang
David T
DT
Chiu
Wah
W
D018835:Chaperonin 10 / Q000737:chemistry / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
D018835:Chaperonin 10; D018834:Chaperonin 60; D018832:Molecular Chaperones