Journal: Nat Protoc / Year: 2022 Title: High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity. Authors: Tao Ni / Thomas Frosio / Luiza Mendonça / Yuewen Sheng / Daniel Clare / Benjamin A Himes / Peijun Zhang / Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below ...Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.
History
Deposition
Jul 29, 2021
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Header (metadata) release
Feb 2, 2022
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Map release
Feb 2, 2022
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Update
Feb 23, 2022
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Current status
Feb 23, 2022
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
EMPIAR-10787 (Title: cryo electron tomography of Apoferritin / Data size: 17.3 Data #1: Unaligned raw tilt-series of apoferritin on graphene grid [tilt series])
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.8 nm / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average electron dose: 102.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Final reconstruction
Applied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity / Number subtomograms used: 4826
Extraction
Number tomograms: 6 / Number images used: 5668
CTF correction
Software - Name: emClarity
Final angle assignment
Type: OTHER / Software - Name: emClarity / Details: cross-correlation
FSC plot (resolution estimation)
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