Tilting the specimen stage provides a simple and generalizable way of increasing the orientation distribution of particles during single-particle cryo-EM data collection. The uniform distribution of particles obtained due to specimen stage tilting enables the improvement of overall map quality and interpretability. Begin by aligning the microscope to ensure parallel illumination of the specimen and minimize coma aberrations.
To identify the squares suitable for data collection, record a grid atlas without stage tilt or with stage tilt, which provides an overview of the overall grid quality and an initial indication of suitable areas for data collection. Inspect the squares manually at the magnification used in square acquisition node. Look for the squares where the foil is intact, does not look dehydrated, and has ideal ice thickness, and move the specimen stage to a square of interest.
Determine the eucentric height for the stage position using an alpha wobbler at around 15 degrees stage tilt and adjust the Z height to bring the stage to eucentric height using the keypad panel for the microscope. Ensure the image shift is minimal during the alpha wobble routine. To find a more accurate Z height, estimate it in the focuser node at the magnification used in square acquisition node.
Adjust the settings for the focuser node and enable or disable the fine Z focus option during the initial queuing of squares. Then press Simulate. Tilt the specimen stage to the desired tilt angle for data collection at the true eucentric height and recenter the stage if necessary.
Press Simulate in the square acquisition node to begin queuing targets for whole acquisition node exposures. Next, select a Z focus target and regions with holes suitable for high magnification exposures and submit the targets to the queue for imaging. Bring the specimen stage back to its untilted state and then move to the next square.
Repeat these steps until an adequate number of whole exposures have been queued. Once all squares are queued, go to the whole targeting node and press Submit Queued Targets. Select Allow for user verification of selected targets in node settings and manually inspect the targets selected by the high magnification exposure acquisition node to test if the automated EM hole finder can accurately identify the suitable regions for image acquisition when the specimen stage is tilted.
Once the user is satisfied with the targeting accuracy, deselect the Allow for user verification of selected targets option for automated data collection. The representative images of the grid at square magnification with different tilt angles collected near and far from the eucentric Z height are shown here. The center of the beam is indicated by the center of the red concentric rings and the green arrow indicates the square of interest.
There is a broken grid feature adjacent to the square of interest for reference. Shown here are the representative hole exposures and 2D class averages collected at 0 degrees, 30 degrees, and 60 degrees tilt angles. Although the protein concentration is unchanged across the different tilt angles, a higher tilt angle makes the imaged area appear more crowded in terms of particle concentration.
2D class averages affected by overcrowding are shown in this red box. The simplicity of the protocol allows any standard user of the microscope to easily adapt a tilted data collection strategy.
