Hybrid Approaches to Structural Characterization of Conformational Ensembles of Complex Macromolecular Systems Combining NMR Residual Dipolar Couplings and Solution X‑ray Scattering

Solving structures or structural ensembles of large macromolecular systems in solution poses a challenging problem. While NMR provides structural information at atomic resolution, increased spectral complexity, chemical shift overlap, and short transverse relaxation times (associated with slow tumbling) render application of the usual techniques that have been so successful for medium sized systems (\u3c50 \u3ekDa) difficult. Solution X-ray scattering, on the other hand, is not limited by molecular weight but only provides low resolution structural information related to the overall shape and size of the system under investigation. Here we review how combining atomic resolution structures of smaller domains with sparse experimental data afforded by NMR residual dipolar couplings (which yield both orientational and shape information) and solution X-ray scattering data in rigid-body simulated annealing calculations provides a powerful approach for investigating the structural aspects of conformational dynamics in large multidomain proteins. The application of this hybrid methodology is illustrated for the 128 kDa dimer of bacterial Enzyme I which exists in a variety of open and closed states that are sampled at various points in the catalytic cycles, and for the capsid protein of the human immunodeficiency virus

Mitochondrial cristae revealed with focused light.

Because of the diffraction resolution barrier, optical microscopes have so far failed in visualizing the mitochondrial cristae, that is, the folds of the inner membrane of this 200 to 400 nm diameter sized tubular organelle. Realizing a ∼30 nm isotropic subdiffraction resolution in isoSTED fluorescence nanoscopy, we have visualized these essential structures in the mitochondria of intact cells. We find a pronounced heterogeneity in the cristae arrangements even within individual mitochondrial tubules

Mechanistic Insight into Nanoparticle Surface Adsorption by Solution NMR Spectroscopy in an Aqueous Gel

Engineering nanoparticle (NP) functions at the molecular level requires a detailed understanding of the dynamic processes occurring at the NP surface. Herein we show that a combination of dark‐state exchange saturation transfer (DEST) and relaxation dispersion (RD) NMR experiments on gel‐stabilized NP samples enables the accurate determination of the kinetics and thermodynamics of adsorption. We used the former approach to describe the interaction of cholic acid (CA) and phenol (PhOH) with ceria NPs with a diameter of approximately 200 nm. Whereas CA formed weak interactions with the NPs, PhOH was tightly bound to the NP surface. Interestingly, we found that the adsorption of PhOH proceeds via an intermediate, weakly bound state in which the small molecule has residual degrees of rotational diffusion. We believe the use of aqueous gels for stabilizing NP samples will increase the applicability of solution NMR methods to the characterization of nanomaterials

WxWindows Interface for CALE

wxWindows is an Open Source, platform independent, User Interface (UI) which has been in development for over eleven years (http://www.wxwindows.org). Currently wxWindows is actively supported for the Linux/Unix (X11, Motif and GTK+), Mac OS 9 and X, all Win32 OSes, MGL, and OS/2 operating systems. wxWindows is written in C++ using an object oriented programming framework; it is a reasonably lightweight API (called wxWidgets) sitting over the native graphics packages of the various platforms it supports. The original version of CALE was written for the basic target platform of Unix using X11 as the graphics package. There have been separate efforts to port the code to Mac OS 9, Mac OS X, Win32, Windows Services for Unix (SFU) and CygWin. Each of these used a variety of different graphical interface approaches and build/make systems. For instance Windows SFU and CygWin could still only use X11 graphics. So could the Win32 version, if a X11 server library and client software were installed. A native Win32 version of CALE was contemplated, but never started. The Macintosh versions were completed but never widely distributed to the users. Given the growing code version support issues, and the slow deviation from the portable code model CALE originally started with, it was desired to come up with a simple graphical UI that would be cross platform portable with only a single code base and build system. During the past two summers, two Laboratory summer students and a CALE team code physicist have worked on porting CALE to the wxWidgets UI. In the summer of 2003 Jeffery Hagelberg (formerly Purdue University, now at the University of California-Davis) started the project. During the spring & summer of 2004 Christopher Egner (Rochester Institute of Technology) completed the work. Paul Amala (A/X-Program at LLNL) supervised the students for their combined 30 weeks of effort. This poster session describes the wxWindows interface as it is implemented in CALE, the level of cross platform portable it actually affords, and the lessons learned during the porting of an existing X11 program to this open source software package. (U

Drift estimation in sparse sequential dynamic imaging, with application to nanoscale fluorescence microscopy.

A major challenge in many modern superresolution fluorescence microscopy techniques at the nanoscale lies in the correct alignment of long sequences of sparse but spatially and temporally highly resolved images. This is caused by the temporal drift of the protein structure, e.g. due to temporal thermal inhomogeneity of the object of interest or its supporting area during the observation process. We develop a simple semiparametric model for drift correction in single-marker switching microscopy. Then we propose an M-estimator for the drift and show its asymptotic normality. This is used to correct the final image and it is shown that this purely statistical method is competitive with state of the art calibration techniques which require the incorporation of fiducial markers in the specimen. Moreover, a simple bootstrap algorithm allows us to quantify the precision of the drift estimate and its effect on the final image estimation. We argue that purely statistical drift correction is even more robust than fiducial tracking, rendering the latter superfluous in many applications. The practicability of our method is demonstrated by a simulation study and by a single-marker switching application. This serves as a prototype for many other typical imaging techniques where sparse observations with high temporal resolution are blurred by motion of the object to be reconstructed

Ground-layer wavefront reconstruction from multiple natural guide stars

Observational tests of ground layer wavefront recovery have been made in open loop using a constellation of four natural guide stars at the 1.55 m Kuiper telescope in Arizona. Such tests explore the effectiveness of wide-field seeing improvement by correction of low-lying atmospheric turbulence with ground-layer adaptive optics (GLAO). The wavefronts from the four stars were measured simultaneously on a Shack-Hartmann wavefront sensor (WFS). The WFS placed a 5 x 5 array of square subapertures across the pupil of the telescope, allowing for wavefront reconstruction up to the fifth radial Zernike order. We find that the wavefront aberration in each star can be roughly halved by subtracting the average of the wavefronts from the other three stars. Wavefront correction on this basis leads to a reduction in width of the seeing-limited stellar image by up to a factor of 3, with image sharpening effective from the visible to near infrared wavelengths over a field of at least 2 arc minutes. We conclude that GLAO correction will be a valuable tool that can increase resolution and spectrographic throughput across a broad range of seeing-limited observations.Comment: 25 pages, 8 figures, to be published in Astrophys.