Spot-scan imaging of microcrystals of an influenza neuraminidase-antibody fragment complex.
Electron micrographs of two-dimensional microcrystals of a complex of an avian influenza virus neuraminidase and an antibody Fab fragment, termed 32/3, have been recorded using the spot-scan method of imaging. The crystals have a large unit cell (159.5 A x 159.5 A x 130.5 A) and a high solvent content (approximately 71% by volume) and are a challenging specimen for testing the spot-scan methodology. Crystalline order was preserved to beyond 4 A resolution as demonstrated by electron diffraction, using an embedding medium of a mixture of glucose and neutral potassium phosphotungstate. Using a Philips C400 computer control system interfaced to an EM420 electron microscope, and with the inclusion of additional software in the system, we have been able to record micrographs at low temperature with a relatively narrow (1500 A diameter) moving beam. There is evidence that the use of such a spot-scan beam reduces the effects of beam-induced specimen motion on the quality of micrographs. Conventional low-dose "flood-beam" images showed good isotropic optical diffraction in only 15% of cases whereas 30% of spot-scan images showed good diffraction. The best flood-beam images gave phases to only 15 A resolution after computer processing, whereas the best spot-scan images gave phases to 7 A resolution. Electron diffraction patterns were also recorded at low temperature, and the resulting diffraction amplitudes combined with phases from spot-scan images to yield a projection map of the structure. A 7 A resolution projection map of the complex is presented, and is compared with the projection map of the same avian influenza neuraminidase complexed with a different monoclonal Fab fragment, NC41, which has been solved to high resolution by X-ray diffraction.
Bullough
P A
PA
MRC Laboratory of Molecular Biology, Cambridge, UK.
Tulloch
P A
PA
D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry*; D001665:Binding Sites; D003460:Crystallization; D003461:Crystallography; D007091:Image Processing, Computer-Assisted; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000648:ultrastructure*; D009980:Influenza A virus / Q000201:enzymology / Q000276:immunology / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009439:Neuraminidase / Q000737:chemistry / Q000648:ultrastructure*; D014961:X-Ray Diffraction
D000914:Antibodies, Viral; D007140:Immunoglobulin Fab Fragments; D009439:Neuraminidase