Automated cryoEM data acquisition and analysis of 284742 particles of GroEL.
One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.
Stagg
Scott M
SM
The National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Lander
Gabriel C
GC
Pulokas
James
J
Fellmann
Denis
D
Cheng
Anchi
A
Quispe
Joel D
JD
Mallick
Satya P
SP
Avila
Radomir M
RM
Carragher
Bridget
B
Potter
Clinton S
CS
D018834:Chaperonin 60 / Q000737:chemistry*; D020285:Cryoelectron Microscopy / Q000379:methods*; D004601:Elementary Particles; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry; D007053:Ice / Q000032:analysis; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008958:Models, Molecular; D012680:Sensitivity and Specificity
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D007053:Ice