Computational protocol: Volta phase plate cryo-EM of the small protein complex Prx3

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Protocol publication

[…] For the first data set, 44 movies were manually collected on a Titan Krios electron microscope (FEI) operated at 300 kV and equipped with a K2 Summit direct detector, a quantum energy filter (Gatan) and an FEI phase plate (FEI). Movies comprising 10 frames and a total dose of 20 e− per Å2 were recorded on a K2 Summit direct detection camera (Gatan) at a calibrated magnification of 37,000 corresponding to a magnified pixel size of 1.35 Å.The recorded movies showed obvious signs of drift and were subjected to combined global and local movement correction by optical flow analysis. One micrograph was subjected to e2boxer in EMAN2 (ref. ) for initial particle picking. The particles were extracted in Relion using a box size of 152 pixels. This small data set comprising 500 particles was used for reference-free two-dimensional (2D) classification in Relion with 10 classes. Of those 10 classes, three classes were deemed suitable to represent the three dominant views—top view, side view and tilted view. These three classes served as templates for automatic particle picking in Relion yielding 30,000 particles. 2D classification of the full data set was used to remove particles that were either false positives or belonged to classes featuring touching particles resulting in a data set of 10,000 particles. The reduced data set was subjected to three-dimensional (3D) classification using the crystal structure of bovine Prx3 dodecamers as initial model after low-pass filtering it to 60 Å, which produced a featureless toroid. 3D classification was used to group the images into three 3D classes imposing D6 symmetry. 7,000 particles were assigned to the most prominent 3D class, which was further processed with auto-refinement in Relion. This yielded a map, which was sharpened in Relion using a negative B-factor of 800. For resolution determination, the so-called ‘gold-standard' Fourier shell correlation (FSC)=0.143 criterion was used. 3D visualizations of the map and rigid-body dockings were carried out in UCSF Chimera.For the second data set, 700 micrographs were recorded on a Titan Krios electron microscope operated at 300 kV and equipped with a K2 Summit direct detector, a quantum energy filter (Gatan) and an FEI phase plate (FEI). Automatic data collection was carried out with SerialEM software. Images were recorded in movie mode comprising 15 frames with a total dose of 20 e− per Å2 at a magnification of 28,700 and a pixel size of 1.74 Å. The frames were aligned as described by Li et al.. Particles (354,000) were semi-automatically picked using EMAN2 (ref. ) e2boxer software and extracted in Relion 1.3 with a box size of 152. False positives were removed by three rounds of 2D classification followed by four rounds of 3D classification. 8,580 particles from the best looking 3D class were subjected to 3D auto-refinement in Relion. Particle-based beam-induced motion correction was performed as part of the movie processing. The produced map was then sharpened in Relion with automatically estimated B-factor of −137. The data collection parameters and refinement statistics for the second data set are summarized in . […]

Pipeline specifications

Software tools EMAN, RELION, UCSF Chimera, SerialEM
Application cryo-EM
Organisms Homo sapiens