**Grid Preparation** 1) All sample preparation steps are performed in accordance to conventional single-particle cryo-EM analysis.
 {"action": "perform", "output": ""}
At the time of writing, gold grids have provided superior results,

any grid substrate that minimizes beam-induced movement can be employed in the future.

For the most part, vitrification is identical between holey carbon gold grids, although some differences might be observed, most notably that blotting times tend to be shorter for gold grids.
{"action": "do", "output": "", "container": ["gold grids"]}
We have also observed slight differences in particle concentration of preferred particle orientation on grids, both of which are specimen dependent.
{"action": "observe", "output": "", "volume": [""]}
**Data Collection** This utilizes the latest Leginon system for data collection. #####%%%%% output "data collection" are used in line14
{"action": "enable", "output": "data collection", "reagent": [""]}
See troubleshooting section for other variations. #####%%%%% output "other variations" are used in line30
{"action": "see", "output": "other variations", "reagent": ["data collection"]}
3) When grid is in microscope, obtain correct eucentric height for the square.
 {"action": "obtain", "output": "", "device": ["microscope"]}
4) Tilt the stage to desired tilt angle by changing the stage alpha parameter.
{"action": "tilt", "output": ""}
5) Acquire low-magnification square image holes, at tilt.

6) Collect high-magnification images of the queued up locations at the specified tilt angle.

See troubleshooting section for alternative approaches to data collection.

**Data Processing** 7) Data processing can be performed conventionally, as per general single-particle cryo-EM procedures.

8) Per-particle CTF estimation must be performed in order to account for the gradient of defocus values present within each micrograph taken at a tilt angle.

Per-particle estimation of CTF can be performed using "GCTF":http://www.mrc-lmb.cam.ac.uk/kzhang/. #####%%%%% output "CTF" are not used
{"action": "perform", "output": "CTF", "reagent": ["other variations"]}
Examples of the commands can be found in the GCTF user guide.

9) 2D classification, can be performed as per conventional processing in all software packages using per-particle defocus values as input.

**Results Analysis** 10) Euler angle distribution serves as a direct evaluation of the degree of preferred orientation \(still) present.
{"action": "provide", "output": "", "reagent": [""]}
11) For a more quantitive directional resolution anisotropy assessment, 3DFSC can be used.

3DFSC can be downloaded from "here":https://github.com/nysbc/Anisotropy.
