Journal: Nat Methods / Year: 2022 Title: Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution. Authors: Ivan Lazić / Maarten Wirix / Max Leo Leidl / Felix de Haas / Daniel Mann / Maximilian Beckers / Evgeniya V Pechnikova / Knut Müller-Caspary / Ricardo Egoavil / Eric G T Bosch / Carsten Sachse / Abstract: In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently ...In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.
Name: RNA (5'-R(P*GP*AP*A)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)
Organism: Tobacco mosaic virus (vulgare)
Molecular weight
Theoretical: 958.66 Da
Sequence
String:
GAA
-
Experimental details
-
Structure determination
Method
cryo EM
Processing
helical reconstruction
Aggregation state
helical array
-
Sample preparation
Concentration
90 mg/mL
Buffer
pH: 7.4
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: blot force of +10 and a duration for blotting of 10 seconds.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Details
iDPC-STEM
Image recording
Film or detector model: OTHER / Average electron dose: 35.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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