Mu DNA transposition proceeds through a series of higher-order nucleoprotein complexes called transpososomes. The structural core of the transpososome is a tetramer of the transposase, Mu A, bound to the two transposon ends. High-resolution structural analysis of the intact transposase and the transpososome has not been successful to date. Here we report the structure of Mu A at 16-Å and the Type 1 transpososome at 34-Å resolution, by 3D reconstruction of images obtained by scanning transmission electron microscopy (STEM) at cryo-temperatures. Electron spectroscopic imaging (ESI) of the DNA-phosphorus was performed in conjunction with the structural investigation to derive the path of the DNA through the transpososome and to define the DNA-binding surface in the transposase. Our model of the transpososome fits well with the accumulated biochemical literature for this intricate transposition system, and lays a structural foundation for biochemical function, including catalysis in trans and the complex circuit of macromolecular interactions underlying Mu DNA transposition.[Keywords: DNA transposition; transpososome; transposase; site-specific recombination; 3D reconstruction; electron spectroscopic imaging; scanning transmission electron microscopy] Supplemental material is available at http://www.genesdev.org. A variety of DNA processes including replication, recombination, transcription, and transposition make use of higher-order nucleoprotein complexes (Echols 1986). These complexes are built by multiple proteins binding cooperatively at multiple DNA sites with the assistance of accessory proteins, which bend DNA to facilitate interaction of proteins bound at distant sites. The requirement for the assembly of such elaborate structures ensures high levels of specificity and a means of providing elaborate regulatory mechanisms.Mu DNA was the first mobile element for which an in vitro transposition system (Mizuuchi 1983) was available (for a recent review, see Chaconas and Harshey 2002) and knowledge gained from this system has proven invaluable for more recent analyses of other transposable elements. Interestingly, the Mu system has also turned out to be one of the most complex and elaborate transposition systems studied to date. The Mu strand transfer reaction, the first stage in the transposition process, requires four proteins: Mu A and Mu B encoded by the transposon and HU and IHF from the host cell. Subsequent steps to complete the replicative transposition process of Mu require a variety of host replication proteins.The Mu A protein active site cleaves at the Mu-host junctions and promotes transesterification of the free 3ЈOH Mu ends to a target DNA molecule. An acidic, metal coordinating DDE motif is a crucial element of the Mu active site, as for other transposons (Mizuuchi and Baker 2002). The Mu B protein captures the target DNA, recruits it to the active site of Mu A, and allosterically activates Mu A for the transesterification step. The Escherichia coli HU and IHF proteins are accessory factors that ...