Introduction

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.

Publications

  1. Automated cryoEM data acquisition and analysis of 284742 particles of GroEL.
    Cite this
    Stagg SM, Lander GC, Pulokas J, Fellmann D, Cheng A, Quispe JD, Mallick SP, Avila RM, Carragher B, Potter CS, 2006-09-01 - Journal of structural biology
  2. Automated molecular microscopy: the new Leginon system.
    Cite this
    Suloway C, Pulokas J, Fellmann D, Cheng A, Guerra F, Quispe J, Stagg S, Potter CS, Carragher B, 2005-07-01 - Journal of structural biology
  3. Rapid routine structure determination of macromolecular assemblies using electron microscopy: current progress and further challenges.
    Cite this
    Carragher B, Fellmann D, Guerra F, Milligan RA, Mouche F, Pulokas J, Sheehan B, Quispe J, Suloway C, Zhu Y, Potter CS, 2004-01-01 - Journal of synchrotron radiation
  4. A relational database for cryoEM: experience at one year and 50 000 images.
    Cite this
    Fellmann D, Pulokas J, Milligan RA, Carragher B, Potter CS, 2002-03-01 - Journal of structural biology
  5. Leginon: an automated system for acquisition of images from vitreous ice specimens.
    Cite this
    Carragher B, Kisseberth N, Kriegman D, Milligan RA, Potter CS, Pulokas J, Reilein A, 2000-10-01 - Journal of structural biology
  6. Leginon: a system for fully automated acquisition of 1000 electron micrographs a day.
    Cite this
    Potter CS, Chu H, Frey B, Green C, Kisseberth N, Madden TJ, Miller KL, Nahrstedt K, Pulokas J, Reilein A, Tcheng D, Weber D, Carragher B, 1999-07-01 - Ultramicroscopy

Credits

  1. Scott M Stagg
    Developer

  2. Gabriel C Lander
    Developer

  3. James Pulokas
    Developer

  4. Denis Fellmann
    Developer

  5. Anchi Cheng
    Developer

  6. Joel D Quispe
    Developer

  7. Satya P Mallick
    Developer

  8. Radomir M Avila
    Developer

  9. Bridget Carragher
    Developer

  10. Clinton S Potter
    Investigator

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Summary
AccessionBT000442
Tool TypeApplication
Category
PlatformsLinux/Unix
TechnologiesC
User InterfaceTerminal Command Line
Download Count0
Submitted ByClinton S Potter