Axel T. Brunger

Crystallography & NMR system: A new software suite for macromolecular structure determination.

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 structure determination by X-ray crystallography and solution NMR.

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 Å resolution of a core synaptic fusion complex containing syntaxin-1A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four α-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Crystallographic R Factor Refinement by Molecular Dynamics

Molecular dynamics was used to refine macromolecular structures by incorporating the difference between the observed crystallographic structure factor amplitude and that calculated from an assumed atomic model into the total energy of the system. The method has a radius of convergence that is larger than that of conventional restrained least-squares refinement. Test cases showed that the need for manual corrections during refinement of macromolecular crystal structures is reduced. In crambin, the dynamics calculation moved residues that were misplaced by more than 3 angstroms into the correct positions without human intervention.

Version 1.2 of the Crystallography and NMR system

Version 1.2 of the software system, termed Crystallography and NMR system (CNS), for crystallographic and NMR structure determination has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure determination, (ii) to provide tools for structure solution of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromolecules and (iv) to integrate all sources of information into all stages of the structure determination process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallographic refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, especially at lower resolution, generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron density maps. Consistent with the modular design of CNS, these additions and changes were implemented in the high-level computing language of CNS.

Slow-cooling protocols for crystallographic refinement by simulated annealing

An improved protocol for crystallographic refinement by simulated annealing is presented. It consists of slow cooling starting at high temperatures. Tests of refinements of aspartate aminotransferase and procin pepsin show that the slow-cooling protocol produces lower R factors and better geometry than other protocols previously published. The influence of the temperature-control method, weighting, cooling rate and duration of the heating stage on the success of the slow-cooling protocol is studied. Analysis of the time course of the potential-energy fluctuations indicates no global changes in the state of order of the system. Fluctuations of the potential energy are interpreted as localized conformational changes during the course of the refinement.

Stochastic boundary conditions for molecular dynamics simulations of ST2 water

Abstract The deformable stochastic boundary method developed previously for treating simple liquids without periodic boundary conditions, is extended to the ST2 model of water. The method is illustrated by a molecular dynamics simulation of a sphere containing 98 water molecules. Comparison with the results of the periodic boundary simulation by Stillinger and Rahman shows very good agreement for structural and dynamic properties.

Assessment of Phase Accuracy by Cross Validation: the Free R Value. Methods and Applications

Analogies between the free R statistic [Brunger (1992). Nature (London), 355, 472–474] and the statistical methods of cross validation and bootstrap are discussed. Several new applications which make use of the previously observed correlation between the free R value and the phase accuracy of crystal structures are presented. One application concerns the relative weighting of individual restraint classes in macromolecular refinement. The free R value provides an objective statistical basis for the optimal choice of the weights. The results for the refinement of a penicillopepsin crystal structure at 1.8 A resolution indicate that overall bond length and bond angle weights, derived from uncertainties observed in small-molecule crystal structures, appear to be transferable to macromolecules. In another application, the landscape of the R value around the crystal structure was investigated for unrestrained modeling of diffraction data with equal atomic scatterers. Others have suggested applications to ab initio phasing because of the simplicity of the liquid-like system of equal atomic scatterers. However, there are a large number of incorrect configurations of the scatterers whose R values at 1.8 A resolution are close to that of the correct configuration given by the positions of the non-hydrogen atoms in the penicillopepsin crystal structure. A substantial number of the incorrect configurations have higher free R values than the correct one. It is therefore conceivable that the free R value could be used as a selection criterion to distinguish between certain incorrect configurations and configurations close to the correct one.

Crystallographic refinement by simulated annealing

Crystallographic refinement by simulated annealing with molecular dynamics has been applied to a 2·8 A (1 A = 0·1 nm) resolution X-ray structure of aspartate aminotransferase. Comparison of the refined structure and a structure obtained by combined restrained least-squares refinement and manual re-fitting shows a similar R factor, stereochemistry, and mean difference from the isomorphous replacement phase centroids. Crystallographic refinement by simulated annealing accomplished structural changes and improvements of the electron density maps that were not possible by using restrained least-squares refinement without manual re-fitting. Crystallographic refinement by simulated annealing can generate an ensemble of structures, each of which agrees with the diffraction information. Regions of large variations of the ensemble indicate either erroneously fitted or disordered segments of the macromolecule.

Interhelical hydrogen bonding drives strong interactions in membrane proteins

Polar residues in transmembrane α-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent miscelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.

Structural Basis of Rab Effector Specificity: Crystal Structure of the Small G Protein Rab3A Complexed with the Effector Domain of Rabphilin-3A

The small G protein Rab3A plays an important role in the regulation of neurotransmitter release. The crystal structure of activated Rab3A/GTP/Mg2+ bound to the effector domain of rabphilin-3A was solved to 2.6 A resolution. Rabphilin-3A contacts Rab3A in two distinct areas. The first interface involves the Rab3A switch I and switch II regions, which are sensitive to the nucleotide-binding state of Rab3A. The second interface consists of a deep pocket in Rab3A that interacts with a SGAWFF structural element of rabphilin-3A. Sequence and structure analysis, and biochemical data suggest that this pocket, or Rab complementarity-determining region (RabCDR), establishes a specific interaction between each Rab protein and its effectors. RabCDRs could be major determinants of effector specificity during vesicle trafficking and fusion.