Characterization of microtubule protofilament numbers.  How does the surface lattice accommodate? 
Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryoelectron microscopy.  Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly.  It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously.  Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions.  Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique.  Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length.  The profiles across the images indicate the protofilament number of the microtubule.  The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist.  In 13 protofilament microtubules the protofilaments are paraxial.  In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis.  The skew is greater for the 15 protofilament case than for 14 protofilaments.  The skew allows the extra protofilaments to be accommodated by the surface lattice.  These results should also be relevant to situations in vivo.