Cell, Vol. 96, 79–88, January 8, 1999, Copyright 1999 by Cell PressHigh-Resolution Model of the MicrotubuleWerecentlyobtainedthestructureofthetubulindimerEva Nogales,*†§Michael Whittaker,‡Ronald A. Milligan,‡and Kenneth H. Downing* by electron crystallography of zinc-induced tubulinsheets (Nogales et al., 1998a). Each tubulin monomer*Lawrence Berkeley National LaboratoryBerkeley, California 94720 (Figure1A) is a compact ellipsoidof approximate dimen-sions 46 3 40 3 65 A˚(width, height, and depth, respec-†Molecular and Cell Biology DepartmentUniversity of California at Berkeley tively) made up of three sequential domains: an N-ter-minal, nucleotide-binding domain; a smaller secondBerkeley, California 94720‡Department of Cell Biology domain; and a predominantly helical C-terminal region.The a and b subunits are very similar, with the ab-dimerThe Scripps Research InstituteLa Jolla, California 92037 being 46 3 80 3 65 A˚. We have also calculated a 20 A˚resolution three-dimensional (3D) map of the microtu-bulebycryoelectronmicroscopyandhelicalreconstruc-tion and established the map polarity by comparisonSummarywith previous data (Sosa and Milligan, 1996; Sosa et al.,1997). The 3D map shows that the inside and outsideA high-resolution model of the microtubule has beenmicrotubule surfaces are very distinctive. The outsideobtained by docking the crystal structure of tubulinsurface is characterized by a shallow zigzag of densityintoa20A˚map of the microtubule. The excellent fitthat forms the crest of the protofilament. In a side view,indicates the similarity of the tubulin conformation inthis surface is fairly flat with very shallow undulations.both polymers and defines the orientation of the tu-In contrast, the inside surface is deeply corrugated, withbulin structure within the microtubule. Long C-termi-the connections between protofilaments lying close tonalhelices form thecrest on theoutside of theprotofil-theinnersurface.Herewehavedocked thecrystalstruc-ament, while long loopsdefine the microtubulelumen.ture of tubulin into the 3D map of the microtubule toThe exchangeable nucleotide in b-tubulin is exposedcreate a near atomic model of the microtubule. Theat the plus end of the microtubule, while the proposedmodel shows the detailed architecture of the microtu-catalytic residue in a-tubulin is exposed at the minusbule and provides insight into the molecular basis forend. Extensive longitudinal interfaces between mono-the observed properties of microtubules.mers have polar and hydrophobic components. At thelateral contacts, a nucleotide-sensitive helix interactswith a loop that contributes to the binding site of taxolResultsin b-tubulin.Microtubule DockingIntroductionAs the high-resolution model was obtained from a poly-merizedform oftubulin (Nogales et al., 1998a), the struc-Microtubules are ubiquitous cytoskeletal elements builtture of the complete protofilament is readily availableby the self-association of ab-tubulin dimers. The poly-from the electron crystallographic data. Thus, dockingmerization process involves two types of contacts be-the high-resolution and low-resolution structures wastween tubulin subunits: head-to-tail binding of dimersgreatly facilitated by fitting the protofilament as a unit.results in protofilaments that run along the length of theFitting was carried out first manually by rotating themicrotubule, and lateral interactions between parallelatomic model of the protofilament around its axis and byprotofilaments complete the microtubule wall. Adjacenttranslating along the microtubulelength. This procedureprotofilaments are offset axially, resulting in a helicalwas repeated for both the up and down orientations oflattice of monomers that is occasionally interrupted bythe protofilament. Only one orientation, rotation, anda “seam” where the lateral interface between protofila-translation fit within the microtubule map, unambigu-ments involves heterologous contacts (a-b) betweenously defining the polarity and orientation of the protofil-monomers. The longitudinal contacts along protofila-amentatomic model (Figures 1B–1D). Subsequently,thements appear to be much stronger than those betweenfitting was quantitatively tested by computing a correla-adjacent protofilaments, based both on the fact thattion between the microtubule reconstruction and a den-depolymerization involves the peeling of protofilamentsity map calculated from the atomic model at a resolu-fragments from the microtubule ends (Mandelkow ettion of 2 A˚. As a function of rotation of the model aboutal., 1991) and on the recurrence of the protofilamentthe protofilament axis, the correlation has a strong peakstructure in all characterized tubulin polymer forms, forthat defines the orientation of the model to within z58example rings, spirals, sheets, or ribbons. Only in micro-(Figure 1E). Such rotation would result in movement attubules and zinc-induced sheets are the protofilamentsthe outer surface of the protofilament of less than 3 A˚.straight.Incontrastwiththemicrotubule,thezincsheetsThe orientation obtained by this method is the same asare formed by the antiparallel association of protofila-that found by the visual docking. Similarly well-definedments (Amos and Baker, 1979).correlation peaks were found for translation along andperpendicular to the axis, defining the position to withinz3A˚(data not shown), although these other degrees§To whom correspondence should be addressed (e-mail: [email protected]).of freedom would not affect the interaction betweenCell80Figure 1. Docking of the Tubulin Crystal Structure into the Microtubule Map(A) Electron crystallographic structure of b-tubulin from zinc-induced sheets stabilized with taxol. The different secondary structure elementsas defined in Nogales et al. (1998a, 1998b) are indicated. Loops involved in nucleotide binding are labeled T1 to T7, where T7 interacts withthe nucleotide of the next tubulin subunit down. The loop marked “M loop” is important in lateral interactions between protofilaments in themicrotubule (see text). Figure generated with Raster 3D (Merrit and Murphy, 1994).(B–E) Docking of the crystal structure of the tubulin protofilament from zinc sheets into the 20 A˚3D map of the microtubule. The microtubulemap was obtained by helical reconstruction of ice-embedded microtubules with 15 protofilaments and a four-start helix as described in Sosaet al. (1997). (B) Front view of the docking shown from the