Journal: Sci Rep / Year: 2021 Title: Below 3 Å structure of apoferritin using a multipurpose TEM with a side entry cryoholder. Authors: Yoko Kayama / Raymond N Burton-Smith / Chihong Song / Naoya Terahara / Takayuki Kato / Kazuyoshi Murata / Abstract: Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA ...Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA are based on data acquired on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment, although this comes with performance limitations. Here, a multi-purpose TEM with a side entry cryo-holder was used to evaluate the potential of high-resolution SPA, resulting in a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. This map clearly showed two positions of an aromatic side chain. Further, examination of optimal imaging conditions depending on two different multi-purpose electron microscope and camera combinations was carried out, demonstrating that higher magnifications are not always necessary or desirable. Since automation is effectively a requirement for large-scale data collection, and augmenting the multi-purpose equipment is possible, we expanded testing by acquiring data with SerialEM using a β-galactosidase test sample. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.
History
Deposition
Mar 12, 2020
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Header (metadata) release
Mar 17, 2021
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Map release
Mar 17, 2021
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Update
Mar 17, 2021
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Current status
Mar 17, 2021
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
JEOL 2200FS
Specialist optics
Energy filter - Name: In-column Omega Filter / Energy filter - Slit width: 15 eV
Image recording
Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.2 mm / Nominal magnification: 100000
Sample stage
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN
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Image processing
CTF correction
Software - Name: CTFFIND (ver. 4.1.10)
Startup model
Type of model: OTHER / Details: Ab initio model generated by cisTEM.
Final reconstruction
Applied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 8762
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)
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