Biomedical subjects

Anchi Cheng

Publications and source records attributed to Anchi Cheng.

9 recordsLinked to original sources

PubMed2006

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.

Cryoelectron Microscopy↗

PubMed2006

Bootstrap resampling for voxel-wise variance analysis of three-dimensional density maps derived by image analysis of two-dimensional crystals.

Difference density maps are commonly used in structural biology for identifying conformational changes in macromolecular complexes. For interpretation of the results, it is essential to estimate the variance or standard deviation of the difference density and the distribution of errors in space. In order to compare three-dimensional density maps of gap junction channels with and without the C-terminal regulatory domain, we developed a bootstrap resampling method for estimation of the voxel-wise standard deviation. The bootstrap approach has been successfully used for estimating the sampling distribution from a limited data set and for estimating the statistical properties of the derived quantities [Efron, B., 1979. Bootstrap methods: another look at the jackknife. Ann. Stat. 7, 1-26]. In our application, the standard deviation map can be estimated by bootstrapping the images. Our results show that, apart from the symmetry axes and small regions bordering the lumen of the extracellular vestibule, difference maps normalized by the mean of the standard deviation map can be used as a good approximation of the t-test map of the gap junction crystals.

Animals↗

PubMed2006

Automated cryoEM data acquisition and analysis of 284742 particles of GroEL.

One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.

Chaperonin 60↗

PubMed2006

Does contamination buildup limit throughput for automated cryoEM?

The development of automated systems for data acquisition in cryo electron microscopy has enabled the possibility of acquiring very large number of images from a single specimen grid. We have demonstrated that over images of 250,000 single particles can be acquired in a 24 h period. This has raised questions as to whether contamination buildup on the specimen limits the quality of the data that can be acquired during these long duration experiments and also whether the data acquisition session could be extended to allow acquisition of more than 1,000,000 particles. We report here a systematic characterization of contamination of specimens maintained for long periods of time at liquid nitrogen temperatures using standard side entry cryo stages. As part of this characterization we developed a more reliable method for accurately estimating specimen ice thickness. Using the method, we were able to calibrate image contrast against ice thickness under a variety of magnifications, objective aperture positions, and defoci, and demonstrated the strong dependence of the calibration curve on these parameters. The results show the anti-contamination aperture is, as expected, critical to the prevention of contamination and that loading film into the microscope dramatically increases the contamination rate, particularly in the first 3 h after the insertion of the film box. In the absence of film, we were able to reproducibly demonstrate that the contamination rate can be limited to a rate of approximately 1 angstrom/h providing reassurance that contamination will not be a major limiting factor for long term cryoEM experiments if a CCD camera is used for the imaging.

Algorithms↗

PubMed2005

Automated molecular microscopy: the new Leginon system.

We report here on the current state of our efforts in automated molecular microscopy. Our primary automated data acquisition software system, Leginon, has been completely redesigned over the past two years. The new distributed system has been developed using the Python programming language and is compatible with both Linux and Windows operating systems. The new flexible architecture was designed to allow for the development of customized data collection protocols, several of which are described here. The system has been used to acquire data for approximately 150 experiments and we have demonstrated the capacity for high throughput data acquisition by acquiring images of more than 100,000 particles in a single session at the microscope.

Microscopy, Electron↗

PubMed2004

A graphical representation of image quality for three-dimensional structure analysis of two-dimensional crystals.

Electron crystallography of two-dimensional (2D) crystals is a powerful approach for the analysis of membrane protein structure. Three-dimensional (3D) structures are derived by merging data from 2D crystals at varying tilt axes and tilt angles. A graphical representation was developed that incorporates the tilt geometry to display the quality of each image. Information that can be extracted from the plot includes a symbol for each image that reflects the completeness of the data for that crystal. The polar plot also includes a novel parameter that reflects the minimal variation in tilt axis to ensure overlap of data points in reciprocal space between images at the same tilt angle. The tilt geometry and image quality plot is especially useful for tracking the progress of data collection and for assessing the completeness of two data sets prior to determination of difference maps.

Connexin 43↗

PubMed2003

Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly.

Although retroviruses from different genera form morphologically distinct capsids, we have proposed that all of these structures are composed of similar hexameric arrays of capsid (CA) protein subunits and that their distinct morphologies reflect different distributions of pentameric declinations that allow the structures to close. Consistent with this model, CA proteins from both HIV-1 and Rous sarcoma virus (RSV) form similar hexagonal lattices. However, recent structural studies have suggested that the Moloney murine leukemia virus (M-MuLV) CA protein may assemble differently. We now report an independent three-dimensional reconstruction of two-dimensional crystals of M-MuLV CA. This new reconstruction reveals a hexameric lattice that is similar to those formed by HIV-1 and RSV CA, supporting a generalized model for retroviral capsid assembly.

Animals↗

PubMed2002

The architecture of a water-selective pore in the lipid bilayer visualized by electron crystallography in vitreous ice.

The water-selective pathway through aquaporin 1 (AQP1) membrane channel has been visualized by fitting an atomic model to a 3.7 A resolution three-dimensional density map. This map was determined by analysing images and electron diffraction patterns of lipid-reconstituted two-dimensional crystals of AQP1 preserved in vitrified buffer in the absence of any additive. The aqueous pathway in a monomer is characterized by a size-selective, to approximately 4.0 +/- 0.5 A wide pore that spans a length of to approximately 18 and bends by to approximately 25 degrees as it traverses the bilayer. This narrow pore is connected by wide, funnel-shaped openings at the extracellular and cytoplasmic faces, and is outlined mostly by hydrophobic residues interspersed with short stretches of polar amino acids, which results in relatively inert pathway conducive to diffusion-limited water flow. Although not visible at the current resolution, the 3D structure suggests putative binding sites for water molecules in the size-selective pore.

Amino Acid Sequence↗

PubMedDate not supplied

Projection structure of full length connexin 43 by electron cryo-crystallography.

We previously used electron cryo-crystallography to determine the three-dimensional structure of recombinant gap junction channels formed by a C-terminal truncation mutant of Cx43 (11). The dodecameric channel is formed by the end-to-end docking of two hexameric connexons, each comprised of 24 transmembrane alpha-helices. We have now generated two-dimensional crystals of the recombinant, full-length channel, as well as crystals in which the C-tail has been completely removed by trypsin digestion. Projection density maps at 7.5 A resolution closely resemble our previous analysis of the C-terminal truncation mutant (9). A difference map between the full length and trypsin-treated channels suggests that there are small but significant shifts in protein density upon removal of the C-tail.

Animals↗