High-resolution cryo-EM reconstructions in the presence of substantial aberrations

Bromberg, Raquel; Guo, Yirui; Borek, Dominika; Otwinowski, Zbyszek

doi:10.1107/s2052252520002444

Cited by 10 publications

(8 citation statements)

References 35 publications

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“…Considering the cost of instrument time, beam-image shift provides 1752 movies per 24 h period versus 720 movies per 24 h period for stage positioning. Indeed, the latest generation of detectors with faster readout stand to triple this throughput for beam-image shift (Alewijnse et al, 2017;Bromberg et al, 2020).…”

Section: Data Throughput Versus Data Qualitymentioning

confidence: 99%

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

Cash

Kearns

et al. 2020

IUCrJ

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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.

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Section: Data Throughput Versus Data Qualitymentioning

confidence: 99%

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

Cash

Kearns

et al. 2020

IUCrJ

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“…Additionally, M detects and corrects for optical aberrations within the data such as astigmatism, anisotropic pixel size, and higher-order aberrations. The improvement in resolution from correcting these aberrations has previously been established in SPA pipelines [78][79][80]. The combination of CTF-related features in the Warp/M packages show impressive improvements in obtainable resolution [37,55].…”

Section: Ctf Correctionmentioning

confidence: 94%

Current data processing strategies for cryo-electron tomography and subtomogram averaging

Pyle

Zanetti

2021

Biochemical Journal

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Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

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“…We solved its Xray crystallographic structure in 2004 (PDB code: 1T0T.pdb), while others determined its function later (Dailey & Gerdes, 2015, Milazzo et al, 2019, Pfanzagl et al, 2018. We recently characterized this protein using cryoEM SPR (EMPIAR-10363, EMD-21373 and EMPIAR-10362, EMD-21376) (Bromberg et al, 2020) and noticed during cryoEM experiments that batches of the protein purified at different times showed different patterns of preferred orientation during cryoEM SPR data collection.…”

Section: Methodsmentioning

confidence: 99%

“…The data were acquired and analyzed as described before (Bromberg et al, 2020). (Baker et al, 2001) to which we provided an input generated with 6VSA.pdb and the server http://server.poissonboltzmann.org/ (Dolinsky et al, 2004).…”

Section: 2mentioning

confidence: 99%

The His-tag as a decoy modulating preferred orientation in cryoEM

Bromberg

Guo

Plymire

et al. 2020

Preprint

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The His-tag is a widely used affinity tag that facilitates purification of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the water-air interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. The analysis of particle orientations suggests that His-tag presence can mask the hydrophobic patches on a protein′s surface that mediate the interactions with the water-air interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with water-air interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the water-air interface.

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High-resolution cryo-EM reconstructions in the presence of substantial aberrations

Cited by 10 publications

References 35 publications

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

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

Current data processing strategies for cryo-electron tomography and subtomogram averaging

The His-tag as a decoy modulating preferred orientation in cryoEM

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