Leginon: New features and applications

Abstract

Leginon is a system for automated data acquisition from a transmission electron microscope. Here we provide an updated summary of the overall Leginon architecture and an update of the current state of the package. We also highlight a few recent developments to provide some concrete examples and use cases.

Keywords: automation; cryoEM; microED.

FIGURE 1

Multi‐Scale Imaging and Leginon accessory functions: Images at higher magnification are acquired by defining targets on the parent images. Acquisition and Targeting node classes string these processes together. Additional targets can be added to perform other automated tasks. Focusing targets (blue) are used to adjust eucentric height and perform low‐dose focusing. Reference targets (violet) are used to perform periodic alignments such as the energy filter zero‐loss slit. The reference node may also pass data to an accessory node to do additional tasks such as ice thickness measurement

FIGURE 2

Leginon architecture: The microscope, cameras and other instruments (phase plates, energy filters, etc.) are controlled by facility staff or local users through the Leginon user interface (see Figure 7). Database and File Servers store the data and metadata which is viewed using a web browser; Appion also uses the web viewer as a user interface for live preprocessing (see Figure 3). A new web interface was recently added to provide remote manual targeting and on‐site support requests (see Figure 8)

FIGURE 3

Web viewers: (a) Integrated Leginon and Appion web tools provide real‐time display of acquired images and results from on‐the‐fly particle picking, CTF estimation, motion correction and ice thickness. (b) Similar tools are available for observing and monitoring microED data collection

FIGURE 4

Summary report: Data summarized includes (a) image statistics; (b) ice thickness; (c) defocus and CTF estimates; (d) overall grid atlas; (e) experimental setup; (f) experimental methods; (g) image processing results uploaded by the operator

FIGURE 5

Leginon microED workflow: First, an atlas is collected to locate good grid squares with crystals. The user then selects grid squares and areas of interest within grid squares for higher magnification images. These images are fed into a queue of images for user targeting. The user can then target suitable crystals from these images and add them to the exposure collection queue. The user has the option to test the diffraction of any crystal with Diffraction Preview Mode. This immediately takes a single diffraction image from the targeted crystal so that the user can decide whether to add that crystal to the queue or omit it based on a visual inspection of the diffraction pattern displayed in the Web Viewer (Figure 3b). Once a sufficient number of crystals have been targeted, the user submits the queue. Leginon then automatically moves to each crystal target, and performs eucentric focusing. The stage is then set to the starting tilt angle and rotated at a constant speed to the desired end point while the camera records diffraction data in rolling shutter mode. The image files are saved, and converted to MRC format, for the web viewer, and SMV format for import into standard crystallographic software. This process repeats until all crystal targets have been processed. Note that the data collection parameters can be altered by the user at any time during the execution of the queue of crystal targets

FIGURE 6

Ice thickness monitoring: Plot of predicted hole ice thickness versus measured high magnification target ice thickness indicates close correlation. To obtain ice thickness between 20 and 80 nm, the hole thickness values should be constrained to values from 0.05 to 0.14 or more precise values can be calculated from the fit

FIGURE 7

Leginon GUI interface: (a) Workflow list and node selection. (b) Toolbar for the selected node in (a), showing Exposure_Targeting in this case. (c) Continuous log of events including warning and errors. (d) Node information panel, in this case providing image statistics. (e) Auto‐targeting types and settings arranged by processing steps

FIGURE 8

Leginon‐remote interface: (a) interactive target selection; (b) Leginon operation status showing nodes requiring remote intervention and nodes that are busy; (c) remote tools available include: grid atlas refresh, queue submission, pause/resume, and column valve closing; (d) session progress; (e) live chat with staff (need some targets on this image)

FIGURE 9

(a) Main interface of tiltgroupwrangler.py. Using the star file provided by Leginon as input, various tools allow examination and modification of the information; a slider is used to select the number of groups to determine. (b) Plot of the tilt groups found; raw tilt values are displayed as blue dots, centroids of each group as red dots, and grouping is visualized by the map colors

FIGURE 10

Annual microscope checkout results: (a) Isosurface representation of the apoferritin map using 325,636 particles from 564 movies taken on a TFS Titan Krios with Gatan GIF/K3 at 0.825 Å/pixel and 54e⁻/Ų total dose. (b) Representative 2D class averages; box size 27.4 × 27.4 nm. (c) An α‐helical segment from one β subunit (PDB: 6V21) is shown in heteroatom representation docked into the corresponding region of the reconstruction. (d) FSC Plot. (e) Guinier Plot