Biomedical subjects

Randy J Read

Publications and source records attributed to Randy J Read.

15 recordsLinked to original sources

PubMed2006

Mutations in FRMD7, a newly identified member of the FERM family, cause X-linked idiopathic congenital nystagmus.

Idiopathic congenital nystagmus is characterized by involuntary, periodic, predominantly horizontal oscillations of both eyes. We identified 22 mutations in FRMD7 in 26 families with X-linked idiopathic congenital nystagmus. Screening of 42 singleton cases of idiopathic congenital nystagmus (28 male, 14 females) yielded three mutations (7%). We found restricted expression of FRMD7 in human embryonic brain and developing neural retina, suggesting a specific role in the control of eye movement and gaze stability.

Brain↗

PubMed2006

Structural mechanism for the carriage and release of thyroxine in the blood.

The hormones that most directly control tissue activities in health and disease are delivered by two noninhibitory members of the serpin family of protease inhibitors, thyroxine-binding globulin (TBG) and corticosteroid-binding globulin. The structure of TBG bound to tetra-iodo thyroxine, solved here at 2.8 A, shows how the thyroxine is carried in a surface pocket on the molecule. This unexpected binding site is confirmed by mutations associated with a loss of hormone binding in both TBG and also homologously in corticosteroid-binding globulin. TBG strikingly differs from other serpins in having the upper half of its main beta-sheet fully opened, so its reactive center peptide loop can readily move in and out of the sheet to give an equilibrated binding and release of thyroxine. The entry of the loop triggers a conformational change, with a linked contraction of the binding pocket and release of the bound thyroxine. The ready reversibility of this change is due to the unique presence in the reactive loop of TBG of a proline that impedes the full and irreversible entry of the loop that occurs in other serpins. Thus, TBG has adapted the serpin inhibitory mechanism to give a reversible flip-flop transition, from a high-affinity to a low-affinity form. The complexity and ready triggering of this conformational mechanism strongly indicates that TBG has evolved to allow a modulated and targeted delivery of thyroxine to the tissues.

Amino Acid Sequence↗

PubMed2006

Crystal structure of rab11 in complex with rab11 family interacting protein 2.

The small GTPase Rab11 regulates the recycling of endosomes to the plasma membrane via interactions with the Rab11 family of interacting proteins (FIPs). FIPs contain a highly conserved Rab binding domain (RBD) at their C termini whose structure is unknown. Here, we have determined the crystal structure of the RBD of FIP2 in complex with Rab11(GTP) by single wavelength anomalous diffraction methods. The overall structure is a heterotetramer with dyad symmetry, arranged as a Rab11-(FIP2)2-Rab11 complex. FIP2 forms a central alpha-helical coiled coil, with both helices contributing to the Rab11 binding patch on equivalent and opposite sides of the homodimer. Switch 1 of Rab11 is embedded between the two helices, while switch 2 remains flexible and is peripherally associated with the effector. The complex reveals the structural basis for Rab11 recognition by FIPs and suggests the molecular mechanisms underlying endocytic recycling pathways.

Amino Acid Sequence↗

PubMed2005

Likelihood-enhanced fast translation functions.

This paper is a companion to a recent paper on fast rotation functions [Storoni et al. (2004), Acta Cryst. D60, 432-438], which showed how a Taylor-series expansion of the maximum-likelihood rotation function leads to improved likelihood-enhanced fast rotation functions. In a similar manner, it is shown here how linear and quadratic Taylor-series expansions and least-squares approximations of the maximum-likelihood translation function lead to likelihood-enhanced translation functions, which can be calculated by FFT and which are more sensitive to the correct translation than the traditional correlation-coefficient fast translation function. These likelihood-enhanced translation targets for molecular-replacement searches have been implemented in the program Phaser using the Computational Crystallography Toolbox (cctbx).

Bacterial Proteins↗

PubMed2005

Liberating crystallographers.

In this issue of Structure, DePristo et al. describe a new program, RAPPER, that should automate some of the most time-consuming tasks associated with rebuilding, refining, and completing protein crystal structures at moderate resolutions.

Crystallography, X-Ray↗

PubMed2004

Solution structure of the Kaposi's sarcoma-associated herpesvirus K3 N-terminal domain reveals a Novel E2-binding C4HC3-type RING domain.

RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys(63)-linked ubiquitination.

Amino Acid Sequence↗

PubMed2004

Simple algorithm for a maximum-likelihood SAD function.

Recently, the multivariate complex normal distribution has been used to develop a maximum-likelihood probability function for single-wavelength anomalous diffraction phasing and refinement of heavy-atom parameters [Pannu & Read (2004), Acta Cryst. D60, 22-27]. The function accounts explicitly for the correlations between the observed and calculated Friedel mates and their errors. However, the method of derivation of the equation described by Pannu & Read (2004) leads to a complicated likelihood expression that suffers from a number of algorithmic limitations. Here, an alternative derivation of the P(SAD) function is described that leads to simplified algorithmic requirements and that allows an intuitive understanding of the expression.

Algorithms↗

PubMed2004

Likelihood-enhanced fast rotation functions.

Experiences with the molecular-replacement program Beast have shown that maximum-likelihood rotation targets are more sensitive to the correct orientation than traditional targets. However, this comes at a high computational cost: brute-force rotation searches can take hours or even days of computation time on current desktop computers. Series approximations to the full likelihood target have been developed that can be computed by fast Fourier transforms in minutes. These likelihood-enhanced targets are more sensitive to the correct orientation than the Crowther fast rotation function and they take advantage of information from partial solutions. The likelihood-enhanced rotation targets have been implemented in the program Phaser.

Algorithms↗

PubMed2003

The application of multivariate statistical techniques improves single-wavelength anomalous diffraction phasing.

Recently, there has been a resurgence in phasing using the single-wavelength anomalous diffraction (SAD) experiment; data from a single wavelength in combination with techniques such as density modification have been used to solve macromolecular structures, even with a very small anomalous signal. Here, a formulation for SAD phasing and refinement employing multivariate statistical techniques is presented. The equation developed accounts explicitly for the correlations among the observed and calculated Friedel mates in a SAD experiment. The correlated SAD equation has been implemented and test cases performed on real diffraction data have revealed better results compared with currently used programs in terms of correlation with the final map and obtaining more reliable phase probability statistics.

Crystallography, X-Ray↗

PubMed2003

Recent developments in the PHENIX software for automated crystallographic structure determination.

A new software system called PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) is being developed for the automation of crystallographic structure solution. This will provide the necessary algorithms to proceed from reduced intensity data to a refined molecular model, and facilitate structure solution for both the novice and expert crystallographer. Here, the features of PHENIXare reviewed and the recent advances in infrastructure and algorithms are briefly described.

Algorithms↗

PubMed2003

New ways of looking at experimental phasing.

In the original work by Blow and Crick, experimental phasing was formulated as a least-squares problem. For good data on good derivatives this approach works reasonably well, but we now attempt to extract more information from poorer data than in the past. As in many other crystallographic problems, the assumptions underlying the use of least squares for phasing are not satisfied, particularly for poor derivatives. The introduction of maximum likelihood (and more powerful computers) has led to substantial improvements. For computational convenience, these new methods still make many assumptions about the independence of different measurements and sources of error. A more general formulation for the probability distributions underlying likelihood-based methods for both experimental phasing and molecular-replacement phasing is reviewed. In the new formulation, all the structure factors associated with a particular hkl are considered to be related by a complex multivariate normal distribution. When it is assumed that certain errors are independent, the general formulation reduces to current likelihood targets. However, the new formulation makes the necessary assumptions more explicit and points the way to improving phasing using both isomorphous and anomalous differences.

Crystallography, X-Ray↗

PubMed2003

Application of the complex multivariate normal distribution to crystallographic methods with insights into multiple isomorphous replacement phasing.

Probabilistic methods involving maximum-likelihood parameter estimation have become a powerful tool in computational crystallography. At the centre of these methods are the relevant probability distributions. Here, equations are developed based on the complex multivariate normal distribution that generalize the distributions currently used in maximum-likelihood model and heavy-atom refinement. In this treatment, the effects of various sources of error in the experiment are considered separately and allowance is made for correlations among sources of error. The multivariate distributions presented are closely related to the distributions previously derived in ab initio phasing and can be applied to many different aspects of a crystallographic structure-determination process including model refinement, density modification, heavy-atom phasing and refinement or combinations of them. The underlying probability distributions for multiple isomorphous replacement are re-examined using these techniques. The re-analysis requires the underlying assumptions to be made explicitly and results in a variance term that, unlike those previously used for maximum-likelihood multiple isomorphous replacement phasing, is expressed explicitly in terms of structure-factor covariances. Test cases presented show that the newly derived multiple isomorphous replacement likelihood functions perform satisfactorily compared with currently used programs.

Crystallography, X-Ray↗

PubMed2003

How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration.

The interaction of the plasma protein vitronectin with plasminogen activator inhibitor-1 (PAI-1) is central to human health. Vitronectin binding extends the lifetime of active PAI-1, which controls hemostasis by inhibiting fibrinolysis and has also been implicated in angiogenesis. The PAI-1-vitronectin binding interaction also affects cell adhesion and motility. For these reasons, elevated PAI-1 activities are associated both with coronary thrombosis and with a poor prognosis in many cancers. Here we show the crystal structure at a resolution of 2.3 A of the complex of the somatomedin B domain of vitronectin with PAI-1. The structure of the complex explains how vitronectin binds to and stabilizes the active conformation of PAI-1. It also explains the tissue effects of PAI-1, as PAI-1 competes for and sterically blocks the interaction of vitronectin with cell surface receptors and integrins. Structural understanding of the essential biological roles of the interaction between PAI-1 and vitronectin opens the prospect of specifically designed blocking agents for the prevention of thrombosis and treatment of cancer.

Amino Acid Sequence↗

PubMed2002

PHENIX: building new software for automated crystallographic structure determination.

Structural genomics seeks to expand rapidly the number of protein structures in order to extract the maximum amount of information from genomic sequence databases. The advent of several large-scale projects worldwide leads to many new challenges in the field of crystallographic macromolecular structure determination. A novel software package called PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) is therefore being developed. This new software will provide the necessary algorithms to proceed from reduced intensity data to a refined molecular model and to facilitate structure solution for both the novice and expert crystallographer.

Algorithms↗

PubMed2002

Crystal structure of double helical hexitol nucleic acids.

A huge variety of chemically modified oligonucleotide derivatives has been synthesized for possible antisense applications. One such derivative, hexitol nucleic acid (HNA), is a DNA analogue containing the standard nucleoside bases, but with a phosphorylated 1',5'-anhydrohexitol backbone. Hexitol nucleic acids are some of the strongest hybridizing antisense compounds presently known, but HNA duplexes are even more stable. We present here the first high-resolution structure of a double helical nucleic acid with all sugars being hexitols. Although designed to have a restricted conformational flexibility, the hexitol oligomer h(GTGTACAC) is able to crystallize in two different double helical conformations. Both structures display a high x-displacement, normal Watson-Crick base pairing, similar base stacking patterns, and a very deep major groove together with a minor groove with increased hydrophobicity. One of the conformations displays a major groove which is wide enough to accommodate a second HNA double helix resulting in the formation of a double helix of HNA double helices. Both structures show most similarities with the A-type helical structure, the anhydrohexitol chair conformation thereby acting as a good mimic for the furanose C3'-endo conformation observed in RNA. As compared to the quasi-linear structure of homo-DNA, the axial position of the base in HNA allows efficient base stacking and hence double helix formation.

DNA↗