The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 Å crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar b-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

Crystallization has proven to be the most significant bottleneck to high-throughput protein structure determination using diffraction methods. We have used the large-scale, systematically generated experimental results of the Northeast Structural Genomics Consortium to characterize the biophysical properties that control protein crystallization. Datamining of crystallization results combined with explicit folding studies lead to the conclusion that crystallization propensity is controlled primarily by the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. These analyses identify specific sequence features correlating with crystallization propensity that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the bulk amino acid sequence properties of human versus eubacterial proteins that reflect likely differences in their biophysical properties including crystallization propensity. Finally, our thermodynamic measurements enable critical evaluation of previous claims regarding correlations between protein stability and bulk sequence properties, which generally are not supported by our dataset. NIH Public Access Author ManuscriptNat Biotechnol. Author manuscript; available in PMC 2010 January 1. Published in final edited form as:Nat Biotechnol. 2009 January ; 27(1): 51-57. doi:10.1038/nbt.1514. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe ability to determine the atomic structures of macromolecules represents a great achievement in molecular biology because of the unparalleled value of this information in understanding the fundamental chemistry of life [1][2][3][4][5] . While nuclear magnetic resonance represents an invaluable source of structural information, especially for small proteins, most macromolecular structures are determined using x-ray crystallography. Capitalizing on the recent proliferation of genomic sequence data, "structural genomics" consortia have been organized worldwide to develop methods and infrastructure for high-throughput protein structure determination. These groups have contributed to improvements in expression and structure determination methods 6 , and the four largest U.S. consortia accounted for 45% of all novel structures deposited in the Protein Data Bank (PDB) in 2007 7 . While these efforts contribute to the impressive progress of the structural biology community in characterizing the full repertoire of protein structures, the rate of growth in sequence information nonetheless far out-paces that of structural information. Given the ongoing acceleration of whole-genome sequencing, the gap between the two will continue to expand without a breakthrough in macromolecular structure determination methods.The systematic efforts of structural genomics projects show that crystallization is the major bottleneck to protein structure determinati...

Patient motion during an MRI exam can result in major degradation of image quality, and is of increasing concern due to the aging population and its associated diseases. This work presents a general strategy for real-time, intraimage compensation of rigidbody motion that is compatible with multiple imaging sequences. Image quality improvements are established for structural brain MRI acquired during volunteer motion. A headband integrated with three active markers is secured to the forehead. Prospective correction is achieved by interleaving a rapid track-and-update module into the imaging sequence. For every repetition of this module, a short tracking pulse-sequence remeasures the marker positions; during head motion, the rigid-body transformation that realigns the markers to their initial positions is fed back to adaptively update the image-plane-maintaining it at a fixed orientation relative to the head-before the next imaging segment of k-space is acquired. In cases of extreme motion, corrupted lines of kspace are rejected and reacquired with the updated geometry. Patient movement is a fundamental problem in virtually all in vivo MR applications. Motion induces local field variations, causes erroneous positional encoding of k-space data, and corrupts the spin-excitation history between slices; these phenomena manifest in image-space as misregistrations, blurring, and ghosting. Even a few millimeters of movement during scanning can produce severe artifacts in reconstructed data, thus rendering images unusable. Often, it is subject populations with the highest potential diagnostic benefit in which the utility of MRI is curtailed by motion artifacts. In a study of 17 patients with frontoparietal tumors, data from five had to be rejected due to gross motion artifacts (1). Even among a healthy elderly population, our experience suggests that significant artifacts may appear in 10% to 20% of high-resolution structural brain scans; typically used for diagnostic and morphological analysis, such scans are especially prone to motion artifact due to their longer duration. To address these concerns, a motion-correction strategy for brain MRI is presented.The fact that the head is a rigid-body (to a very close approximation) allows an arbitrary motion to be described by six degrees-of-freedom (6-DOF)-three rotations about a three-dimensional (3D) orthogonal coordinate-system, and three translations. Retrospective motion-compensation methods, such as those used to coregister multiple image volumes in functional MRI (fMRI) studies, are well established. The most popular algorithms (2) determine the 6-DOF via minimization of a least-squares cost function and only correct for interimage motion. Retrospective correction involves interpolation, which can cause image blurring, and is further limited by its inability to fully correct for the influences of through-plane motion on local spin-history.In contrast, prospective strategies compensate for motion in the acquisition stage by keeping the image-plane at a fixed orientation ...