RESUMEN
In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to â¼3 Å). Using this approach, we determined a 3.3-Å-resolution structure of an â¼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency-key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.
Asunto(s)
Microscopía por Crioelectrón/métodos , Complejo de la Endopetidasa Proteasomal/ultraestructura , Thermoplasma/enzimología , Electrones , Imagenología Tridimensional/métodos , Movimiento (Física)RESUMEN
Dual-axis electron microscopic tomography minimizes the missing wedge-induced resolution loss by taking two complementary tilt data sets of the same target along two orthogonal axes. The potential of this powerful approach has been hampered by the practical challenges inherent in finding the original targets that are dramatically displaced due to non-eucentric specimen rotation. Not only is the manual search for the original targets time consuming and tedious but the added dose during manual searching is uncontrollable. We have developed a hierarchical alignment scheme that allows tomographic data to be collected from an arbitrary number of target sites in one grid orientation and then to find and collect orthogonal data sets with little or no user intervention. Inspired by the successful multi-scale mapping in Leginon, our alignment is performed in three levels to gradually pinpoint the original targets. At the lowest level the grid lattice is used to determine the rotation angle and translational shift resulting from specimen rotation via auto- and cross-correlative analysis of a pair of atlas maps constructed before and after specimen rotation. The target locations are further refined at the next level using a pair of smaller atlas maps. The final refinement of target positions is done by aligning the target contained image tiles. Given the batch processing nature of this hierarchical alignment, multiple targets are initially selected in a group and then sequentially acquired. Upon completion of the data collection on all the targets along the first axis and after specimen rotation, the hierarchical alignment is performed to relocate the original targets. The data collection is then resumed on these targets for the second axis. Therefore, only one specimen rotation is needed for collecting multiple dual-axis tomographic data sets. The experiment of acquiring 20S Proteasomes dual-axis tomographic data sets in vitreous ice at 86,000x CCD magnification on our FEI Tecnai Polara TF30 electron microscope has suggested that the developed scheme is very robust. The extra doses for finding and centering the original targets are almost negligible. This scheme has been integrated into UCSF Tomography software suite that can be downloaded at www.msg.ucsf.edu/tomography free for academic use.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodosRESUMEN
The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1-2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase-specific inhibitor of SPB maturation whose expression is under control of cdc10(+). This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fase G1 , Fase S , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Fase G1/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Hidroxiurea/farmacología , Microscopía Electrónica , Nitrógeno/deficiencia , Nitrógeno/metabolismo , Fase S/efectos de los fármacos , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructura , Factores de Tiempo , Factores de TranscripciónRESUMEN
Full resolution electron microscopic tomographic (EMT) reconstruction of large-scale tilt series requires significant computing power. The desire to perform multiple cycles of iterative reconstruction and realignment dramatically increases the pressing need to improve reconstruction performance. This has motivated us to develop a distributed multi-GPU (graphics processing unit) system to provide the required computing power for rapid constrained, iterative reconstructions of very large three-dimensional (3D) volumes. The participating GPUs reconstruct segments of the volume in parallel, and subsequently, the segments are assembled to form the complete 3D volume. Owing to its power and versatility, the CUDA (NVIDIA, USA) platform was selected for GPU implementation of the EMT reconstruction. For a system containing 10 GPUs provided by 5 GTX295 cards, 10 cycles of SIRT reconstruction for a tomogram of 4096(2) × 512 voxels from an input tilt series containing 122 projection images of 4096(2) pixels (single precision float) takes a total of 1845 s of which 1032 s are for computation with the remainder being the system overhead. The same system takes only 39 s total to reconstruct 1024(2) × 256 voxels from 122 1024(2) pixel projections. While the system overhead is non-trivial, performance analysis indicates that adding extra GPUs to the system would lead to steadily enhanced overall performance. Therefore, this system can be easily expanded to generate superior computing power for very large tomographic reconstructions and especially to empower iterative cycles of reconstruction and realignment.
Asunto(s)
Tomografía con Microscopio Electrónico/estadística & datos numéricos , Algoritmos , Animales , Centrosoma/ultraestructura , Redes de Comunicación de Computadores , Gráficos por Computador , Drosophila/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/estadística & datos numéricosRESUMEN
Single particle reconstruction using the random conical tilt data collection geometry is a robust method for the initial determination of macromolecular structures by electron microscopy. Unfortunately, the broad adoption of this powerful approach has been limited by the practical challenges inherent in manual data collection of the required pairs of matching high and low tilt images (typically 60 degrees and 0 degrees). The microscopist is obliged to keep the imaging area centered during tilting as well as to maintain accurate focus in the tilted image while minimizing the overall electron dose, a challenging and time consuming process. To help solve these problems, we have developed an automated system for the rapid acquisition of accurately aligned and focused tilt pairs. The system has been designed to minimize the dose incurred during alignment and focusing, making it useful in both negative stain and cryo-electron microscopy. The system includes a feature for montaging untilted images to ensure that all of the particles in the tilted image may be used in the reconstruction.
Asunto(s)
Microscopía por Crioelectrón/métodos , Imagenología Tridimensional/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Diseño de Software , Biología Computacional , Escherichia coli , Ribosomas/químicaRESUMEN
We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed that the 30-40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on their topology and high degree of interconnectedness, it is unlikely that 30-40-nm domains arise from the folding of local stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments. By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network, while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30-40-nm chromatin domains. These observations suggest that 30-40-nm domains are essential for establishing long-range chromatin associations that are central for chromosome condensation.
Asunto(s)
Adenosina Trifosfatasas/análisis , Cromatina/ultraestructura , Cromosomas/ultraestructura , ADN-Topoisomerasas de Tipo II/análisis , Proteínas de Unión al ADN/análisis , Complejos Multiproteicos/análisis , Animales , Cromatina/química , Microscopía Electrónica , Modelos Moleculares , Tomografía , Xenopus laevisRESUMEN
A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.
Asunto(s)
Recolección de Datos/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica/métodos , Programas Informáticos , Algoritmos , Cromosomas Humanos/química , Sistemas de Computación , Células HeLa , Humanos , Diseño de SoftwareRESUMEN
Incorporation of a block copolymer into a thin polymer film is observed to alter both the rate and mechanism by which the film dewets from an immiscible polymer substrate. Films with little or no copolymer dewet by classical nucleation and growth of circular holes, and the dewetting rate decreases with increasing copolymer concentration. Increasing the copolymer content at constant film thickness generates copolymer micelles that adsorb/aggregate along the polymer/polymer interface and promote nonclassical dewetting fluctuations similar in appearance to spinodal dewetting. At higher copolymer concentrations, dewetting proceeds after a lengthy induction period by the nucleation and growth of flower-shaped holes suggestive of film pinning or viscous fingering. Atomic force microscopy of the polymer/polymer interface after removal of the top film by selective dissolution reveals substantial structural development due to copolymer self-assembly.
Asunto(s)
Polimetil Metacrilato/química , Poliestirenos/química , Microscopía de Fuerza AtómicaRESUMEN
The amplitude contrast of frozen-hydrated biological samples was measured using the bacterial flagellar filament embedded in vitreous ice at an accelerating voltage of 300kV. From the mean radial amplitude spectra of overfocused images, amplitude contrast was estimated to be 6.9+/-1.9% and 2.7+/-1.0% of the whole contrast at the low spatial frequency range with and without energy filtering, respectively, and that of the carbon film to be 9.5+/-2.0% and 5.8+/-1.8%. Energy filtering effectively doubled the signal-to-noise ratio in the images of frozen-hydrated filaments, and substantially improved intensity data statistics of layer lines up to at least approximately 25A resolution in their Fourier transforms. It also markedly improved inter-particle fitting phase residuals of averaged data at resolutions up to approximately 15A. Using the energy filtered data recorded on a new high-performance, lens-coupled CCD camera the three-dimensional map of the flagellar filament was calculated at 8A by applying the amplitude contrast of 6.9%. The map and its mean radial density distribution validated the obtained value of the amplitude contrast.
Asunto(s)
Microscopía por Crioelectrón/métodos , Aumento de la Imagen/métodos , Proteínas/ultraestructura , Algoritmos , Proteínas Bacterianas/ultraestructura , Flagelos/ultraestructura , Flagelina/ultraestructura , Reproducibilidad de los Resultados , Salmonella/metabolismo , Salmonella/ultraestructuraRESUMEN
A prediction-based scheme is proposed and implemented for automated electron microscopic tomography. By assuming that the sample follows a simple geometric rotation and that the optical system can be characterized in terms of an offset between the optical and mechanical axes, it is found that the image movement in the x, y, and z directions due to stage tilt can be dynamically predicted with desired accuracy (15 nm in x-y position and 100 nm in focus). Thus, the microscope optical system (beam/image shift and focus) can be automatically adjusted to compensate for the predicted image movement prior to taking the projected image at each tilt angle. As a consequence, it is not necessary to either record additional images for tracking and focusing during the course of data collections or to spend valuable setup time in a lengthy pre-calibration of stage motions. Furthermore, this scheme is also found to tolerate a significant degree of non-eucentricity and to be quite robust in the collection of regular and cryo low-dose images on thin or thick samples even at magnifications greater than 62000x and angular step as large as 10 degrees. For interested users the software can be freely downloaded for non-profit use at http://www.msg.ucsf.edu/tomography.