Automated image acquisition for single-particle reconstruction using p97 as the biological sample.
We have used Leginon, a fully automatic system capable of acquiring cryo-electron micrographs, to collect data of single particles, specifically of the AAA ATPase p97. The images were acquired under low-dose conditions and required no operator intervention other than the initial setup and periodic refilling of the cold-stage dewar. Each image was acquired at two different defocus values. Two-dimensional projection maps of p97 were calculated from these data and compared to results previously obtained using the conventional manual data collection methods to film. The results demonstrate that Leginon performs as well as an experienced microscopist for the acquisition of single-particle data. The general advantages of automation are discussed.
Rouiller
I
I
Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
Pulokas
J
J
Butel
V M
VM
Milligan
R A
RA
Wilson-Kubalek
E M
EM
Potter
C S
CS
Carragher
B O
BO
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods* / Q000639:trends; D001330:Electronic Data Processing; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D009687:Nuclear Proteins / Q000737:chemistry*; D010316:Particle Size; D011487:Protein Conformation
D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C404942:p97 ATPase