4.6A Cryo-EM reconstruction of tobacco mosaic virus from images recorded at 300 keV on a 4k x 4k CCD camera.
ABSTRACT: Tobacco mosaic virus (TMV) is a plant virus with a highly ordered organisation and has been described in three different structural states: As stacked disks without RNA (X-ray crystallography), as a helical form with RNA (X-ray fibre diffraction) and as a second distinct helical form with RNA (cryo-EM). Here we present a structural analysis of TMV as a test object to assess the quality of cryo-EM images recorded at 300 keV on a CCD camera. The 4.6A TMV structure obtained is consistent with the previous cryo-EM structure and confirms that there is a second helical form of TMV. The structure here also shows that with a similar number of TMV segments an equivalent resolution can be achieved with a 4k CCD camera at 300 keV.
Project description:Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300?keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200?keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200?keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9?Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8?Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200?keV.
Project description:Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
Project description:Carbon ion radiotherapy is a sophisticated radiation treatment modality because of its superiority in achieving precise dosage distribution and high biological effectiveness. However, there exist beam range uncertainties that affect treatment efficiency. This problem can be resolved if the clinical beam could be monitored precisely in real-time, such as by imaging the prompt gamma emission from the target. In this study, we performed real-time detection and imaging of 718 keV prompt gamma emissions using a Si/CdTe Compton camera. We conducted experiments on graphite phantoms using clinical carbon ion beams of 290 MeV/u energy. Compton images were reconstructed using simple back-projection methods from the energy events of 718 keV prompt gamma emissions. The peak intensity position in reconstructed 718 keV prompt gamma images was few millimeters below the Bragg peak position. Moreover, the dual- and triple-energy window images for all positions of phantoms were not affected by scattered gammas, and their peak intensity positions were approximately similar to those observed in the reconstructed 718 keV prompt gamma images. In conclusion, the findings of the current study demonstrate the feasibility of using our Compton camera for real-time beam monitoring of carbon ion beams under clinical beam intensity.
Project description:An essential step in 2D DIGE-based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge-coupled device (CCD) camera-based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, high-end laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection.
Project description:100?kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200?keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300?Å in thickness, the predicted optimal electron energy for imaging is 100?keV. Currently available electron cryomicroscopes in the 100-120?keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100?keV electrons. To demonstrate the potential of imaging at 100?kV, the voltage of a standard, commercial 200?kV field-emission gun (FEG) microscope was reduced to 100?kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100?keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5?MDa to 64?kDa with corresponding diameters from 320 to 72?Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4?Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100?keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.
Project description:With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.
Project description:Background:Ca2+ plays an important role in many physiological processes and an accurate study of these signals is important. In modern fluorescence microscopy, a charge-coupled device (CCD) camera is widely deployed for calcium imaging. The ratiometric method is used for the fluorescence dye Fura-2 and Grynkiewitz's formula (Grynkiewicz et al., 1985) is commonly used to convert fluorescence to free Ca2+ concentration ([Ca2+]). But the need to subtract the background signal can lead to a big error in ratiometric calcium measurements. When the error due to background subtraction occurs, the fluorescence ratio of 340 nm divided by 380 nm lights may be twice as large as the actual value. Under conditions when the excitation intensity is not adjusted to ensure the same throughput of the objective lens for ultraviolet dye illumination, the indicator does not gradually bleach out for channels with a wavelength of 340 nm and 380 nm light, which lead to an additional error in determining the concentration of Ca2+. New method:Here we present a new approach for calculating [Ca2+] from the ratiometric fluorescence of Fura-2 dye imaged by a CCD camera. It is designed to optimize [Ca2+] measurements with photobleaching correction without background subtraction error. A mathematical method is also provided for removing the existing underestimated value of fluorescence at an excitation wavelength of 340 nm and compensating for the bleaching rate for both channels with wavelengths of 340 nm and 380 nm using a power function. Results:In cultured neurons, the calculations of the free Ca2+ concentration during Ca2+ transients estimated by the old and new methods, determine it to the same extent. This comparison was made under conditions without errors through background subtraction. If there is this error, the old method calculates [Ca2+] with a much higher, rather than the actual value. Conclusions:We present a modified Grynkiewitz's formula for calculation [Ca2+] for ratiometric dye, such as Fura-2 imaged by a CCD camera, with photobleaching correction without background subtraction error.
Project description:Stable capsid structures of viruses protect viral RNA while they also require controlled disassembly for releasing the viral genome in the host cell. A detailed understanding of viral disassembly processes and the involved structural switches is still lacking. This process has been extensively studied using tobacco mosaic virus (TMV), and carboxylate interactions are assumed to play a critical part in this process. Here, we present two cryo-EM structures of the helical TMV assembly at 2.0 and 1.9 Å resolution in conditions of high Ca2+ concentration at low pH and in water. Based on our atomic models, we identify the conformational details of the disassembly switch mechanism: In high Ca2+ /acidic pH environment, the virion is stabilized between neighboring subunits through carboxyl groups E95 and E97 in close proximity to a Ca2+ binding site that is shared between two subunits. Upon increase in pH and lower Ca2+ levels, mutual repulsion of the E95/E97 pair and Ca2+ removal destabilize the network of interactions between adjacent subunits at lower radius and release the switch for viral disassembly.
Project description:Transmission electron microscopy (TEM) of vitrified biological macromolecules (cryo-EM) is limited by the weak phase contrast signal that is available from such samples. Using a phase plate would thus substantially improve the signal-to-noise ratio. We have previously demonstrated the use of a high-power Fabry-Perot cavity as a phase plate for TEM. We now report improvements to our laser cavity that allow us to achieve record continuous wave intensities of over 450 GW/cm<sup>2</sup>, sufficient to produce the optimal 90° phase shift for 300 keV electrons. In addition, we have performed the first cryo-EM reconstruction using a laser phase plate, demonstrating that the stability of this laser phase plate is sufficient for use during standard cryo-EM data collection.
Project description:Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100?kDa using a transmission electron microscope operating at 200?keV coupled with a direct electron detector. Our ~2.7?Å structure of alcohol dehydrogenase (82?kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8?Å and ~3.2?Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64?kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.