Correlative montage parallel array cryo-tomography for in situ structural cell biology

Jie E Yang; Matthew R Larson; Bryan S Sibert; Joseph Y Kim; Daniel Parrell; Juan C Sanchez; Victoria Pappas; Anil Kumar; Kai Cai; Keith Thompson; Elizabeth R Wright

doi:10.1038/s41592-023-01999-5

Correlative montage parallel array cryo-tomography for in situ structural cell biology

Nat Methods. 2023 Oct;20(10):1537-1543. doi: 10.1038/s41592-023-01999-5. Epub 2023 Sep 18.

Authors

Jie E Yang^{1

2

3}, Matthew R Larson^#^{1

2

3}, Bryan S Sibert^#^{1

2

3}, Joseph Y Kim^#^{1

4}, Daniel Parrell^{1

5}, Juan C Sanchez^{1

6}, Victoria Pappas^{1

6}, Anil Kumar^{1

2

3}, Kai Cai^{1

2

3}, Keith Thompson^{1

2

3}, Elizabeth R Wright^{7

8

9

10

11}

Affiliations

¹ Department of Biochemistry, University of Wisconsin, Madison, WI, USA.
² Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA.
³ Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA.
⁴ Department of Chemistry, University of Wisconsin, Madison, WI, USA.
⁵ DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI, USA.
⁶ Biophysics Graduate Program, University of Wisconsin, Madison, WI, USA.
⁷ Department of Biochemistry, University of Wisconsin, Madison, WI, USA. [email protected].
⁸ Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA. [email protected].
⁹ Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA. [email protected].
¹⁰ DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI, USA. [email protected].
¹¹ Morgridge Institute for Research, Madison, WI, USA. [email protected].

^# Contributed equally.

Abstract

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Correlative montage parallel array cryo-tomography for in situ structural cell biology

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