TOMOMAN: Streamlining Cryo-electron Tomography and Subtomogram Averaging Workflows Using TOMOgram MANager

Abstract

Cryo-electron tomography coupled with subtomogram averaging is becoming the preferred tool for investigating cellular and macromolecular structures in their native or near-native environments.Despite this, the process of resolving these structures, starting from the stage of data acquisition to structure determination, is becoming progressively more complex with an increasing number of packages for each step.These packages are developed independently with different design philosophies and input types, making it complicated to interface with one another to form a cohesive pipeline.Tomogram Manager (TOMOMAN) is a MATLAB-based modular package developed to reduce this complexity and streamline the process of resolving structures by subtomogram averaging (Figure 1).TOMOMAN achieves this by maintaining the essential metadata in a MATLAB structure format for all tilt-series in the dataset.The core of TOMOMAN package comprises wrapper scripts for each step of preprocessing from the raw tilt series data to reconstructed tomographic volumes.For motion correction of frames from direct detection devices, TOMOMAN offers wrappers for MotionCor2 and a modified version of RELION's implementation of MotionCor.Resulting tilt stacks are then cleaned in a supervised fashion to eliminate any obvious outliers (contamination, run-away defocus, etc.).For CTF estimation, TOMOMAN offers a choice of CTFFIND4, gCTF, and our own implementation of CTF estimation for tilted data, tiltCTF.For subsequent alignment of these stacks and tomogram reconstruction, TOMOMAN offers a multitude of options to a user.While there are wrappers to streamline the tilt series-alignment using IMOD, we have also implemented wrapper scripts for recently developed AreTomo package for automated tilt series alignment.Reconstruction of uncorrected or CTF corrected tomogram can be performed using wrapper scripts for IMOD or NovaCTF.resulting tomograms and associated metadata are readily exportable for template matching, subtomogram averaging, and classification in STOPGAP.Although, TOMOMAN was initially developed in conjunction with STOPGAP, TOMOMAN projects can also be directly exported to WARP/RELION3/M workflow as well as newly developed RELION4 tomography workflow for subtomogram averaging.Furthermore, there are scripts to import particles from RELION to STOPGAP, facilitating the interoperability.In addition, TOMOMAN also offers a set of wrapper scripts to refine the initial tilt series alignment using either IMOD's local alignments or TomoAlign's motion aware reconstruction by tracking subvolume fiducials in STOPGAP_refine.