Automated three-dimensional reconstruction of keyhole limpet hemocyanin type 1.
We have reconstructed a three-dimensional map of keyhole limpet hemocyanin isoform 1 (KLH1), using our automated data collection software, Leginon, integrated with particle selection algorithms, and the SPIDER reconstruction package. KLH1, a 7.9 MDa macromolecule, is an extracellular respiratory pigment composed of two asymmetric decamers, and presents an overall D(5) point-group symmetry. The reconstruction is in agreement with previous data published on molluscan hemocyanins. The reconstructed map (11.3A resolution, 3sigma criterion) was used to fit an available X-ray crystallography structure of Octopus dofleini Odg, solved at 2.3A [J. Mol. Biol. 278 (4) (1998) 855], with satisfactory results. The results validate the approach of automating the cryoEM process and demonstrate that the quality of the images acquired and the particles selected is comparable to those obtained using manual methods. Several problems remain to be solved however before these results can be generalized.
Mouche
Fabrice
F
Center for Integrative Molecular Biosciences and Department of Cell Biology, The Scripps Research Institute, CB 129, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Zhu
Yuanxin
Y
Pulokas
James
J
Potter
Clinton S
CS
Carragher
Bridget
B
D000465:Algorithms; D000818:Animals; D002138:Calibration; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D019992:Databases as Topic; D019281:Dimerization; D006433:Hemocyanins / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D049831:Octopodiformes; D011487:Protein Conformation; D020033:Protein Isoforms; D012984:Software; D013997:Time Factors; D014961:X-Ray Diffraction
D020033:Protein Isoforms; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin