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TitleRoutine single particle CryoEM sample and grid characterization by tomography.
Journal, issue, pagesElife, Vol. 7, Year 2018
Publish dateMay 29, 2018
AuthorsAlex J Noble / Venkata P Dandey / Hui Wei / Julia Brasch / Jillian Chase / Priyamvada Acharya / Yong Zi Tan / Zhening Zhang / Laura Y Kim / Giovanna Scapin / Micah Rapp / Edward T Eng / William J Rice / Anchi Cheng / Carl J Negro / Lawrence Shapiro / Peter D Kwong / David Jeruzalmi / Amedee des Georges / Clinton S Potter / Bridget Carragher /
PubMed AbstractSingle particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we ...Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.
External linksElife / PubMed:29809143 / PubMed Central
MethodsEM (tomography)
Structure data

EMDB-7135:
Hemagglutinin on a carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7138:
Rabbit muscle aldolase on a gold nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7139:
Rabbit muscle aldolase on a carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7140:
Protein in nanodisc on a gold nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7141:
Glutamate dehydrogenase on a holey carbon grid
Method: EM (tomography)

EMDB-7142:
Glutamate dehydrogenase on a holey carbon grid
Method: EM (tomography)

EMDB-7143:
GDH + 0.001% DDM on a carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7144:
DNAB helicase-helicase loader on a gold Quantifoil grid
Method: EM (tomography)

EMDB-7145:
Apoferritin on a gold nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7146:
Apoferritin on a gold nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7147:
Apoferritin on a holey carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7148:
Apoferritin on a holey carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7149:
Apoferritin on a holey gold nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7150:
Apoferritin with 0.5 mM TCEP on a carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

EMDB-7151:
T20S proteasome on a holey carbon grid
Method: EM (tomography)

EMDB-7152:
T20S proteasome on a holey carbon grid
Method: EM (tomography)

EMDB-7153:
T20S proteasome on a gold Quantifoil grid
Method: EM (tomography)

EMDB-7154:
Mtb 20S proteasome on a carbon nanowire grid plunged with Spotiton
Method: EM (tomography)

Source
  • unidentified (others)
  • Oryctolagus cuniculus (rabbit)
  • Mycobacterium tuberculosis (bacteria)

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