Adopting a database as a solution to managing electron image data.

A database was used for data management and interprogram communication in an image processing and three-dimensional reconstruction program suite for biological bundles. The programs were modified from the MRC crystallographic package. The database server works with local and remote programs and data sets, allows simultaneous requests from multiple clients, and maintains multiple databases and data tables within them. It has built-in security for the data access. Several graphical user interfaces are available to view and/or edit data tables. In addition, FORTRAN interface and function libraries are written to communicate with image processing software. The data management overhead is inexpensive, requiring only narrow bandwidth from the network. It easily handles several data tables with over 1000 entries.

ESPript: analysis of multiple sequence alignments in PostScript.

MOTIVATION: The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS: ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY: ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.

Organisation and structure of microtubules and microtubule-motor protein complexes.

We present a short overview of the current status of work on the organisation and structure of microtubules and of microtubule-motor protein complexes. At present there is great interest in obtaining structural information that can help us to understand the movement of the kinesin family of microtubule associated molecular motors. Using electron cryomicroscopy and image reconstruction methods three dimensional maps of microtubule-motor complexes have been obtained in the presence of different nucleotides. We address a number of principles involved in different aspects of this work.

Tomography without tilt: three-dimensional imaging of microtubule/motor complexes.

Usually structures such as microtubules are supposed to have surface lattices built from families of continuous helices, giving electron micrographs that can be analyzed by helical diffraction theory with a view to obtaining three-dimensional reconstructions. In the case of microtubules the helical surface lattice may be interrupted by discontinuities, called seams, in which case the usual helical reconstruction approach is no longer applicable. Even so, by virtue of their "superhelical" protofilaments, microtubules are still helical structures and we use this feature to treat a microtubule image as a set of projections equivalent to images obtained in a single axis tilt series. The main thrust of this article is to discuss how to obtain images and image parameters best suited to a tomographic approach to three-dimensional reconstruction. The method is tested by comparing helical and back-projection reconstructions of appropriate microtubules both with and without surface lattice decoration by kinesin family motor proteins. Tomographic reconstruction gives an independent demonstration that in vitro assembled microtubules have a B-type surface lattice. We show that 3-start, 15-protofilament microtubules have a seam, whereas 4-start microtubules have no seam and possess complete helical symmetry. Monomer motor domains attach to the outer ridge of the protofilaments and extend along the protofilament toward the plus end.

Three-dimensional structure of functional motor proteins on microtubules.

BACKGROUND: Kinesins are a superfamily of motor proteins that use ATP hydrolysis to fuel movement along microtubules and participate in many crucial phases of the eukaryotic cell cycle. Usually these motors are heterotetramers of two heavy and two light chains, and have globular motor domains on the two heavy chains. Most kinesins move towards the microtubule 'plus end', but some, such as ncd (nonclaret disjunctional protein), move in the opposite direction. Heavy chain dimers produced by overexpression are viable motors. RESULTS: In order to establish whether the opposite directionality of kinesin and ncd dimers is related to notable conformational differences, we have used electron cryo-microscopy and three-dimensional reconstruction methods to investigate the structure of kinesin and ncd dimers attached to microtubules in the presence of AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP analogue. Three-dimensional maps of the motor-microtubule complexes show the motors to have one unattached, and one attached head per tubulin dimer. The polarity of the reconstructions was determined for each individual microtubule. Attachment occurs on the crest of a protofilament at the end of the tubulin dimer that points towards the plus end of the microtubule. The attached head extends over the next tubulin molecule along the protofilament. The unattached heads of kinesin and ncd have distinctly different conformations. CONCLUSIONS: The attached heads of kinesin and ncd appear to be similar and to interact with the same region of the plus end-oriented tubulin subunits. The free heads, however, are quite different, which suggests that directionality could be determined by differences in the dimer conformations. Work is in progress to obtain three-dimensional maps in the presence of different nucleotides with the aim of understanding how these motors move along microtubules.

Lattice defects in microtubules: protofilament numbers vary within individual microtubules.

We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.