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  • The EMBO Journal (2003) 22, 4365 - 4374
  • doi:10.1093/emboj/cdg420

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 Å resolution

Johannes Furst1,2,5, R.Bryan Sutton3,4,5, James Chen1, Axel T. Brunger3 and Nikolaus Grigorieff1

  1. Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA 02454, USA
  2. Department of Physiology, University of Innsbruck, Fritz-Pregl Stras zlige 3, A-6020 Innsbruck, Austria
  3. Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H.Clark Center, E300-C, 318 Campus Drive, Stanford, CA 94305-5432, USA
  4. Present address: Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641, USA
  5. J.Furst and R.B.Sutton contributed equally to this work

Correspondence to:

Axel T. Brunger, E-mail: brunger@stanford.edu

Nikolaus Grigorieff, E-mail: niko@brandeis.edu

Received 25 April 2003; Accepted 7 July 2003; Revised 12 June 2003

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP–ADP mixture at 11 Å resolution by electron cryomicroscopy and single-particle averaging of NSFdotalpha-SNAPdotSNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.

  • Keywords:

    • AAA ATPase,
    • electron cryomicroscopy,
    • FREALIGN,
    • NSF,
    • vesicle fusion