Anchi Cheng

Control of Self-Assembly of DNA Tubules Through Integration of Gold Nanoparticles

The assembly of nanoparticles into three-dimensional (3D) architectures could allow for greater control of the interactions between these particles or with molecules. DNA tubes are known to form through either self-association of multi-helix DNA bundle structures or closing up of 2D DNA tile lattices. By the attachment of single-stranded DNA to gold nanoparticles, nanotubes of various 3D architectures can form, ranging in shape from stacked rings to single spirals, double spirals, and nested spirals. The nanoparticles are active elements that control the preference for specific tube conformations through size-dependent steric repulsion effects. For example, we can control the tube assembly to favor stacked-ring structures using 10-nanometer gold nanoparticles. Electron tomography revealed a left-handed chirality in the spiral tubes, double-wall tube features, and conformational transitions between tubes.

Three-dimensional organization of a human water channel

Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells,. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7Å resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an in-plane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted α-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer.

Structure of Full-Length HIV-1 CA: A Model for the Mature Capsid Lattice

The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids are modeled as fullerene structures composed of closed hexameric arrays of the viral CA protein, but a high-resolution structure of the lattice has remained elusive. A three-dimensional map of two-dimensional crystals of the R18L mutant of HIV-1 CA was derived by electron cryocrystallography. The docking of high-resolution domain structures into the map yielded the first unambiguous model for full-length HIV-1 CA. Three important protein-protein assembly interfaces are required for capsid formation. Each CA hexamer is composed of an inner ring of six N-terminal domains and an outer ring of C-terminal domains that form dimeric linkers connecting neighboring hexamers. Interactions between the two domains of CA further stabilize the hexamer and provide a structural explanation for the mechanism of action of known HIV-1 assembly inhibitors.

An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

Beam-induced motion of vitrified specimen on holey carbon film.

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.

Movies of ice-embedded particles enhance resolution in electron cryo-microscopy.

Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.

Fully automated, sequential tilt-series acquisition with Leginon

Electron tomography has become a uniquely powerful tool for investigating the structures of individual cells, viruses, and macromolecules. Data collection is, however, time consuming and requires expensive instruments. To optimize productivity, we have incorporated one of the existing tilt-series acquisition programs, UCSF Tomo, into the well-developed automatic electron microscopy data collection package Leginon to enable fully automatic, sequential tilt-series acquisition. Here we describe how UCSF Tomo was integrated into Leginon, what users must do to set up a data collection session, how the automatic collection proceeds, how archived data about the process can be accessed and used, and how the software has been tested.

2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy

Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design. DOI: http://dx.doi.org/10.7554/eLife.06380.001

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.

Initial evaluation of a Direct Detection Device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.