Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV

Wu, Mengyu; Lander, Gabriel C.; Herzik, Mark A.

doi:10.1016/j.yjsbx.2020.100020

Cited by 47 publications

(57 citation statements)

References 57 publications

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“…To reach sub-2 Å resolution reconstruction the C s value was refined from the nominal value of 2.7 to 2.55 mm. The deviation of the refined values of C s from the nominal values specified by the manufacturer have also been observed for other microscopes and can indicate specific properties of the particular objective lens as well as incorporate other systematic changes of experimental conditions including altered accelerating voltage (Wu et al, 2020).…”

Section: Benchmark With Apoferritinmentioning

confidence: 66%

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

Fislage

Shkumatov

Stroobants

et al. 2020

Int Union Crystallogr J

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Single-particle cryo-EM has become an indispensable structural biology method. It requires regular access to high-resolution electron cryogenic microscopes. To fully utilize the capacity of the expensive high-resolution instruments, the time used for data acquisition and the rate of data collection have to be maximized. This in turn requires high stability and high uptime of the instrument. One of the first 300 kV JEOL CRYO ARM 300 microscopes has been installed at the cryo-EM facility BECM at VIB-VUB, Brussels, where the microscope is used for continuous data collection on multiple projects. Here, the suitability and performance of the microscope is assessed for high-throughput single-particle data collection. In particular, the properties of the illumination system, the stage stability and ice contamination rates are reported. The microscope was benchmarked using mouse heavy-chain apoferritin which was reconstructed to a resolution of 1.9 Å. Finally, uptime and throughput statistics of the instrument accumulated during the first six months of the facility operation in user access mode are reported.

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Section: Benchmark With Apoferritinmentioning

confidence: 66%

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

Fislage

Shkumatov

Stroobants

et al. 2020

Int Union Crystallogr J

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“…There is some utility in comparison of our results to far superior ones of the same complex obtained elsewhere, such as EMD-9599 (Danev et al, 2019), EMD-9865 (Kato et al, 2019) in the electron microscopy data bank (EMDB), as they were acquired using equipment providing the very highest performance possible for cryo-EM SPA. The 1.75 Å map of apoferritin (Wu et al, 2020) from a 200KV microscope further demonstrates the possibilities with increased quantities of data when coupled to optimal grids, sample preparation technique and microscope stability. Although of far greater value is comparing against other datasets, such as the T20S proteasome (Herzik et al, 2017) where ∼3 Å (2.8 Å highest local resolution) allowed identification of side chain orientation and tightly bound water molecules on a 200kV microscope, albeit one equipped with an autoloader stage and optimised for SPA.…”

Section: Discussionmentioning

confidence: 89%

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

Kayama

Burton‐Smith

Song

et al. 2020

Preprint

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15The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single 16 particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results 17 of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. 18These are currently only available in limited locations around the world, where securing machine 19 time is highly competitive. One potential solution for this time-competitive situation is to reuse 20 existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-21 holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 Å resolution 22 map of apoferritin with local resolution extending to 2.6 Å. The map clearly showed two positions 23 of an aromatic side chain. We also verified the optimal imaging conditions depending on different 24 electron microscope and camera combinations. This study demonstrates the possibilities of more 25 widely available and established electron microscopes, and their applications for cryo-EM SPA. 26 27 Keywords 28 apoferritin; benchmarking; cryo-electron microscopy; multi-purpose TEM; single particle 29 analysis; side-entry cryo-holder 30

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“…Considering the effectiveness of more precise CTF models, including a recently developed algorithm which corrects higher-order aberrations 8,25 , the use of optimal mask diameter and box size is also expected to become increasingly crucial for these algorithms to work.…”

Section: Discussionmentioning

confidence: 99%

“…7Choose the larger of Bf and Bp as the optimal box size Bopt. (8) Execute initial 3D refinement using Bopt for the box size and Pinit for the mask diameter, to get the 3D density map Vinit. (9) In Vinit, measure the diameter of the negative particle density volume -surrounding the positive particle density volume.…”

Section: Proposal Of Optimization Protocolmentioning

confidence: 99%

“…Recently it has been demonstrated that SPA using 200 kV cryo-EM, equipped with a direct electron detector (DED), is capable of reconstructing structures of proteins smaller than 200 kDa at sub-3.0 Å 4,7,8 . The authors resolved ∼150 kDa rabbit muscle aldolase to 2.6 Å and later improved the reconstruction to 2.13 Å using conventional defocus-based SPA methods.…”

Section: Introductionmentioning

confidence: 99%

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Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

Moriya

Adachi

Kawasaki

et al. 2020

Preprint

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Recently it has been demonstrated that single-particle cryogenic electron microscopy (cryo-EM) at 200 keV is capable of determining protein structures, including those smaller than 100 kDa, at sub-3.0 Å resolutions, without using significant defocus or a phase plate. However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 keV. Consequently, many typical parameter settings for the cryo-EM computational image processing steps, especially those associated with the contrast transfer function, are based on the accumulated experience of 300 kV cryo-EM. We have therefore revised these parameters, established theoretical bases for criteria to find an optimal mask diameter and box size for a given dataset irrespective of acceleration voltage or protein size, and proposed a protocol. Considering the defocus distributions of the datasets, merely optimizing the mask diameters and box sizes yielded meaningful resolution improvements for the reconstruction of < 200 kDa proteins using 200 kV cryo-EM.

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Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV

Cited by 47 publications

References 57 publications

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

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Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV

Cited by 47 publications

References 57 publications

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

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Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV