CryoTEM with a Cold Field Emission Gun That Moves Structural Biology into a New Stage

Kato, Takayuki; Makino, Fumiaki; Nakane, Takanori; Terahara, Naoya; Kaneko, Takeshi; Shimizu, Yuko; Motoki, Shinichi; Ishikawa, Isamu; Yonekura, Koji; Namba, Keiichi

doi:10.1017/s1431927619005725

Cited by 51 publications

(42 citation statements)

References 3 publications

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“…Currently, cryo-EM structures with resolutions in the range of 3-4 Å are common 44 . Resolutions in the range of 1.5-2 Å, however, have been restricted to standard benchmark proteins, such as apo-ferritin 22,45 and β-galactosidase 46 , and other highly symmetric samples, including an adeno-associated virus serotype 2 (AAV2) icosahedral capsid variant 47 , the dodecameric urease from Y. enterocolitica 48 and sheep lens connexin 28 .…”

Section: Discussionmentioning

confidence: 99%

Single-particle cryo-EM at atomic resolution

Nakane

Kotecha

Sente

et al. 2020

Preprint

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The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryomicroscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1,2 . However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer 3 . Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. Adrian Koh, Toby Darling and Jake Grimmett for support with computing; Garbi Lezcano Singla, Erik Franken for support with the EER format; and Gerald van Hoften and Gerard Hosmar for support with the Falcon-4 camera.

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Section: Discussionmentioning

confidence: 99%

Single-particle cryo-EM at atomic resolution

Nakane

Kotecha

Sente

et al. 2020

Preprint

Self Cite

123

139

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“…(c) FSC curves: gold standard masked half-maps (black) and map versus model (red). refinement compared with other very high-resolution reconstructions of mouse heavy-chain apoferritin obtained with CRYO ARM 300 (Kato et al, 2019). Nevertheless, despite the low particle density, sub-2 Å resolution reconstruction was still possible.…”

Section: Figurementioning

confidence: 92%

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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“…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.…”

Section: Discussionmentioning

confidence: 86%

“…Developments contributing to advances in SPA have been mainly improvements of electron microscope performance (Knapek et al, 1982; Morishita et al, 2013), developments of electron beam direct detectors (Bammes et al, 2012; McMullan et al, 2016), methods for three-dimensional structure reconstructions (Grant et al, 2018; Kimanius et al, 2016; Punjani et al, 2017; Zivanov et al, 2018), and automated acquisition via Leginon (Carragher et al, 2000), SerialEM (Mastronarde, 2005) or manufacturer software. In recent years, close to atomic resolution has been achieved (Bartesaghi et al, 2015; Danev et al, 2019; Kato et al, 2019) which permits construction of atomic models without foreknowledge of the protein sequence.…”

Section: Introductionmentioning

confidence: 99%

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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CryoTEM with a Cold Field Emission Gun That Moves Structural Biology into a New Stage

Cited by 51 publications

References 3 publications

Single-particle cryo-EM at atomic resolution

Single-particle cryo-EM at atomic resolution

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

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