A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible.The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular (i') 1998 International Union of Crystallography Printed in Great Britain -all rights reserved structure determination by X-ray crystallography and solution NMR.

The ‘tubulin-code’ hypothesis proposes that different tubulin genes or posttranslational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogenous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin “signatures”, which supports the premise of tubulin-code hypothesis.

Microtubules are dynamic polymers of αβ-tubulin that are essential for intracellular organization, organelle trafficking and chromosome segregation. Microtubule growth and shrinkage occur via addition and loss of αβ-tubulin subunits, which are biochemical processes. Dynamic microtubules can also engage in mechanical processes, such as exerting forces by pushing or pulling against a load. Recent advances at the intersection of biochemistry and mechanics have revealed the existence of multiple conformations of αβ-tubulin subunits and their central role in dictating the mechanisms of microtubule dynamics and force generation. It has become apparent that microtubule-associated proteins (MAPs) selectively target specific tubulin conformations to regulate microtubule dynamics, and mechanical forces can also influence microtubule dynamics by altering the balance of tubulin conformations. Importantly, the conformational states of tubulin dimers are likely to be coupled throughout the lattice: the conformation of one dimer can influence the conformation of its nearest neighbours, and this effect can propagate over longer distances. This coupling provides a long-range mechanism by which MAPs and forces can modulate microtubule growth and shrinkage. These findings provide evidence that the interplay between biochemistry and mechanics is essential for the cellular functions of microtubules.

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All TOGether Now αβ-Tubulin is the polymerizing subunit of microtubules, which are dynamic polymers that have essential roles in cell division and intracellular organization. TOG domains are αβ-tubulin binding modules that occur in the evolutionarily conserved Stu2p/XMAP215 family of proteins and promote microtubule elongation. Ayaz et al. (p. 857 ) used crystallographic and biochemical experiments to reveal that the TOG1 domain interacts with guanosine triphosphate–bound αβ-tubulin in a conformation-selective manner, binding preferentially to a “curved,” microtubule-incompatible conformation. The binding mode apparently excludes analogous binding of a second TOG domain to the same heterodimer and may help to ensure polarized growth of microtubules.

A reduced variable conformational sampling strategy for macromolecules based on molecular dynamics in torsion angle space is evaluated using crystallographic refinement as a prototypical search problem. Bae and Haug's algorithm for constrained dynamics [Bae, D.S., Haug, E.J. A recursive formulation for constrained mechanical system dynamics. Mech. Struct. Mach. 15:359-382, 1987], originally developed for robotics, was used. Their formulation solves the equations of motion exactly for arbitrary holonomic constraints, and hence differs from commonly used approximation algorithms. It uses gradients calculated in Cartesian coordinates, and thus also differs from internal coordinate formulations. Molecular dynamics can be carried out at significantly higher temperatures due to the elimination of the high frequency bond and angle vibrations. The sampling strategy presented here combines high temperature torsion angle dynamics with repeated trajectories using different initial velocities. The best solutions can be identified by the free R value, or the R value if experimental phase information is appropriately included in the refinement. Applications to crystallographic refinement. Applications to crystallographic refinement show a significantly increased radius of convergence over conventional techniques. For a test system with diffraction data to 2 A resolution, slow-cooling protocols fail to converge if the backbone atom root mean square (rms) coordinate deviation from the crystal structure is greater than 1.25 A, but torsion angle refinement can correct backbone atom rms coordinate deviations up to approximately 1.7 A.

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, ␣␤-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in ␣␤-tubulin. The allosteric model posits that unpolymerized ␣␤-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of ␣␤-tubulin, the 2.3 Å crystal structure of ␥-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous ␥-tubulin:GTP␥S structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of ␣␤-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive ␣␤-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. ␣␤-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior. dynamic instability ͉ microtubules M icrotubules are hollow cylindrical polymers of ␣␤-tubulin that are critical for intracellular trafficking and formation of the mitotic spindle required for chromosome segregation during cell division. Although vitally important, the molecular mechanisms underlying dynamic microtubule (MT) behavior are poorly understood. Microtubule assembly requires GTP-bound ␣␤-tubulin, and GTP hydrolysis by ␤-tubulin is required to generate dynamic MTs (reviewed in ref. 1). Structural studies have demonstrated two extreme conformations of ␣␤-tubulin: a ''straight'' conformation observed in the MT lattice (2), and a "curved" conformation observed in a structure of ␣␤-tubulin complexed with colchicine and a stathmin-like domain (3) (Fig. 1). There are two opposing models for the relationship between GTP and conformational change in ␣␤-tubulin. The allosteric model (recently reviewed in ref. 4) postulates that GTP binding to ␣␤-tubulin triggers long-range conformational changes yielding a substantially straighter conformation that is prestructured in solution for lateral interactions (5). The lattice model (6) postulates that in solution, ␣␤-tubulin adopts the MTincompatible, curved conformation independent of nuc...

Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully ‘curved’ conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end.DOI: http://dx.doi.org/10.7554/eLife.03069.001