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TitleHigh-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.
Journal, issue, pagesNat Protoc, Vol. 17, Issue 2, Page 421-444, Year 2022
Publish dateJan 12, 2022
AuthorsTao Ni / Thomas Frosio / Luiza Mendonça / Yuewen Sheng / Daniel Clare / Benjamin A Himes / Peijun Zhang /
PubMed AbstractCryo-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.
External linksNat Protoc / PubMed:35022621 / PubMed Central
MethodsEM (subtomogram averaging)
Resolution2.86 - 7.0 Å
Structure data

EMDB-13270:
Subtomogram averaging of E. coli ribosome at 7 angstrom resolution
Method: EM (subtomogram averaging) / Resolution: 7.0 Å

EMDB-13271:
Subtomogram averaging of apoferritin at 2.86 angstrom resolution
Method: EM (subtomogram averaging) / Resolution: 2.86 Å

EMDB-13354:
A 3.3 Angstrom structure of HIV-1 CA-SP1 by subtomogram averaging (5 tilt-series)
Method: EM (subtomogram averaging) / Resolution: 3.3 Å

EMDB-13390:
subtomogram averaging of a HIV-1 immature Gag at 4.5 angstrom resolution
Method: EM (subtomogram averaging) / Resolution: 4.5 Å

Source
  • Escherichia (bacteria)
  • Equus caballus (horse)
  • Human immunodeficiency virus 1

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