Cryo-EM Performance Testing of Hardware and Data Acquisition Strategies

The increasing popularity and adoption rate of cryo-electron microscopy is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the instruments improved dramatically in recent years, but site-specific issues or unnoticed problems during installation could undermine productivity. Newcomers to the field may also have limited experience and/or low confidence in the capabilities of the equipment or their own skills. Therefore, it is recommended to perform an initial test of the complete cryo-EM workflow with an ‘easy’ test sample, such as apoferritin, before starting work with real and challenging samples. Analogous test experiments are also recommended for quantification of new data acquisition approaches or imaging hardware. Here, we present the results from our initial tests of a recently installed Krios G4 electron microscope equipped with two latest generation direct electron detector cameras—Gatan K3 and Falcon 4. Three beam-image shift-based data acquisition strategies were also tested. We detail the methodology and discuss the critical parameters and steps for performance testing. The two cameras performed equally, and the single and multi-shot per-hole acquisition schemes produced comparable results. We also evaluated the effects of environmental factors and optical flaws on data quality. Our results reaffirmed the exceptional performance of the software aberration correction in Relion in dealing with severe coma aberration. We hope that this work will help cryo-EM teams in their testing and troubleshooting of hardware and data collection approaches.

Coma-corrected rapid single-particle cryo-EM data collection on the CRYO ARM 300

Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition of multiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.

Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.

Prevalence and Distribution of the Middle Mesial Canal in Mandibular First Molar Teeth of the Kerman Population: A CBCT Study

Purpose. Missed additional canals are one of the most important reasons for RCT failures in molar teeth. This study aimed to determine the prevalence and distribution of middle mesial canals in mandibular first molars of the Kerman population. Materials and Methods. A retrospective study was performed on de-identified cone beam image sets from 3 private radiology centers in Kerman. A total of 100 mandibular first molars from 62 patients (mean age 32 years) were included. Information regarding the patient’s age and gender, the location of teeth, and the presence or absence of a MMC and also a second distal canal in MMC cases was recorded in an Excel table. Data were analyzed using SPSS software (IBM-USA). Results. The overall prevalence of MMCs in the mandibular first molars was 8.1% (10.0% in females and 6.3% in males). More cases of MMCs were seen on the right side (12.2%) than the left side (3.4%). One case of bilateral MMCs combined with bilateral second distal canals in the mandibular first molars was seen in a 26-year-old female. A further case of bilateral MMCs was found in a 32-year-old male but with single-canal distal roots on both sides. Conclusion. The overall prevalence of MMCs in the Kerman population (8.1%) is at the lower end of the reported range of the international literature (0.26% to 53.8%). In the cohort examined in this study, mid mesial canals were more prevalent in females and on the right side. There was no definite relationship between MMCs and second distal canals in the mandibular first molar teeth in the same subject. MMCs may be unilateral or bilateral. Careful exploration of the pulpal floor between canal orifices is essential to prevent missing the MMC, as this would cause undesirable clinical outcomes.

High-resolution cryo-EM using beam-image shift at 200 keV

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.

Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

ABSTRACTTomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by an order of magnitude and improve map resolution by ~1-3 Å compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.