Bryant Gipson

High-resolution low-dose scanning transmission electron microscopy

During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

Computational Models of Protein Kinematics and Dynamics: Beyond Simulation

Physics-based simulation represents a powerful method for investigating the time-varying behavior of dynamic protein systems at high spatial and temporal resolution. Such simulations, however, can be prohibitively difficult or lengthy for large proteins or when probing the lower-resolution, long-timescale behaviors of proteins generally. Importantly, not all questions about a protein system require full space and time resolution to produce an informative answer. For instance, by avoiding the simulation of uncorrelated, high-frequency atomic movements, a larger, domain-level picture of protein dynamics can be revealed. The purpose of this review is to highlight the growing body of complementary work that goes beyond simulation. In particular, this review focuses on methods that address kinematics and dynamics, as well as those that address larger organizational questions and can quickly yield useful information about the long-timescale behavior of a protein.

Milestones in electron crystallography

Electron crystallography determines the structure of membrane embedded proteins in the two-dimensionally crystallized state by cryo-transmission electron microscopy imaging and computer structure reconstruction. Milestones on the path to the structure are high-level expression, purification of functional protein, reconstitution into two-dimensional lipid membrane crystals, high-resolution imaging, and structure determination by computer image processing. Here we review the current state of these methods. We also created an Internet information exchange platform for electron crystallography, where guidelines for imaging and data processing method are maintained. The server (http://2dx.org) provides the electron crystallography community with a central information exchange platform, which is structured in blog and Wiki form, allowing visitors to add comments or discussions. It currently offers a detailed step-by-step introduction to image processing with the MRC software program. The server is also a repository for the 2dx software package, a user-friendly image processing system for 2D membrane protein crystals.

3D reconstruction from 2D crystal image and diffraction data.

Electron crystallography of 2D protein crystals can determine the structure of membrane embedded proteins at high resolution. Images or electron diffraction patterns are recorded with the electron microscope of the frozen hydrated samples, and the 3D structure of the proteins is then determined by computer data processing. Here we introduce the image-processing algorithms for crystallographic Fourier space based methods using the Medical Research Council (MRC) programs, and illustrate the usage of the software packages 2dx, XDP, and IPLT.

Resolution Independent Density Estimation for motion planning in high-dimensional spaces

This paper presents a new motion planner, Search Tree with Resolution Independent Density Estimation (STRIDE), designed for rapid exploration and path planning in high-dimensional systems (greater than 10). A Geometric Near-neighbor Access Tree (GNAT) is maintained to estimate the sampling density of the configuration space, allowing an implicit, resolution-independent, Voronoi partitioning to provide sampling density estimates, naturally guiding the planner towards unexplored regions of the configuration space. This planner is capable of rapid exploration in the full dimension of the configuration space and, given that a GNAT requires only a valid distance metric, STRIDE is largely parameter-free. Extensive experimental results demonstrate significant dimension-dependent performance improvements over alternative state-of-the-art planners. In particular, high-dimensional systems where the free space is mostly defined by narrow passages were found to yield the greatest performance improvements. Experimental results are shown for both a classical 6-dimensional problem and those for which the dimension incrementally varies from 3 to 27.

SIMS: A Hybrid Method for Rapid Conformational Analysis

Proteins are at the root of many biological functions, often performing complex tasks as the result of large changes in their structure. Describing the exact details of these conformational changes, however, remains a central challenge for computational biology due the enormous computational requirements of the problem. This has engendered the development of a rich variety of useful methods designed to answer specific questions at different levels of spatial, temporal, and energetic resolution. These methods fall largely into two classes: physically accurate, but computationally demanding methods and fast, approximate methods. We introduce here a new hybrid modeling tool, the Structured Intuitive Move Selector (sims), designed to bridge the divide between these two classes, while allowing the benefits of both to be seamlessly integrated into a single framework. This is achieved by applying a modern motion planning algorithm, borrowed from the field of robotics, in tandem with a well-established protein modeling library. sims can combine precise energy calculations with approximate or specialized conformational sampling routines to produce rapid, yet accurate, analysis of the large-scale conformational variability of protein systems. Several key advancements are shown, including the abstract use of generically defined moves (conformational sampling methods) and an expansive probabilistic conformational exploration. We present three example problems that sims is applied to and demonstrate a rapid solution for each. These include the automatic determination of “active” residues for the hinge-based system Cyanovirin-N, exploring conformational changes involving long-range coordinated motion between non-sequential residues in Ribose-Binding Protein, and the rapid discovery of a transient conformational state of Maltose-Binding Protein, previously only determined by Molecular Dynamics. For all cases we provide energetic validations using well-established energy fields, demonstrating this framework as a fast and accurate tool for the analysis of a wide range of protein flexibility problems.

Image processing of 2D crystal images.

Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to image frozen-hydrated 2D crystals. The processing of recorded images exploits the periodic arrangement of the structures in the images to extract the amplitudes and phases of diffraction spots in Fourier space. However, image imperfections require a crystal unbending procedure to be applied to the image before evaluation in Fourier space. We here describe the process of 2D crystal image unbending, using the 2dx software system.

Merging of image data in electron crystallography.

Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to record images and diffraction patterns of frozen-hydrated 2D crystals. Each two-dimensional (2D) crystal is only imaged once, at one specific tilt angle, and the recorded images can be automatically processed with the 2dx/MRC software package. Processed image data from non-tilted and tilted 2D crystals then need to be merged into a 3D reconstruction of the membrane protein structure. We here describe the process of the 3D merging, using the 2dx software system.

Automation of Image Processing in Electron Crystallography

Electron crystallography of membrane proteins records images and diffraction patterns of frozen-hydrated two-dimensional (2D) crystals. To reconstruct the high-resolution three-dimensional (3D) structure of a membrane protein, a multitude of images of 2D crystals have to be processed. Certain processing steps are thereby similar for batches of images that were recorded under similar conditions. Here we describe how the 2dx software package can be used to automate the processing of 2D crystal images, and how the 2D and 3D merging results can be used to iteratively reprocess the images. While the processing of 2D crystal images has been fully automated, the merging process is still semi-manual.

Filling the missing cone: automatic recovery of data in tilt-limited microscopy

Filling the missing cone: Automatic recovery of data in tilt-limited microscopy. Henning Stahlberg, Xiangyan Zeng, Daniel J. Masiel, Nigel Browning, John Spence, Kaoru Mitsuoka, and Bryant Gipson C-CINA, Biozentrum, University Basel, Switzerland, Dep. of Math and Comp. Science, Fort Valley State Univ., GA 31030, USA ,Dep. Chemical Engineering and Mat. Sciences, UC Davis, Davis, CA 95616, USA, Dep. of Physics, Arizona State University, Tempe, AZ 85287, USA, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan E-mail: Henning.Stahlberg@unibas.ch