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TitleBenchmarking the ideal sample thickness in cryo-EM.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 118, Issue 49, Year 2021
Publish dateDec 7, 2021
AuthorsMichael W Martynowycz / Max T B Clabbers / Johan Unge / Johan Hattne / Tamir Gonen /
PubMed AbstractThe relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K ...The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.
External linksProc Natl Acad Sci U S A / PubMed:34873060 / PubMed Central
MethodsEM (electron crystallography)
Resolution1.85 - 2.9 Å
Structure data

EMDB-25456, PDB-7svy:
MicroED structure of proteinase K from a 130 nm thick lamella measured at 120 kV
Method: EM (electron crystallography)

EMDB-25457, PDB-7svz:
MicroED structure of proteinase K from a 200 nm thick lamella measured at 120 kV
Method: EM (electron crystallography)

EMDB-25458, PDB-7sw0:
MicroED structure of proteinase K from a 325 nm thick lamella measured at 120 kV
Method: EM (electron crystallography)

EMDB-25459, PDB-7sw1:
MicroED structure of proteinase K from a 115 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25460, PDB-7sw2:
MicroED structure of proteinase K from a 130 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25461, PDB-7sw3:
MicroED structure of proteinase K from a 95 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25462, PDB-7sw4:
MicroED structure of proteinase K from a 540 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25463, PDB-7sw5:
MicroED structure of proteinase K from a 460 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25464, PDB-7sw6:
MicroED structure of proteinase K from a 260 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25465, PDB-7sw7:
MicroED structure of proteinase K from a 530 nm thick lamella measured at 200 kV
Method: EM (electron crystallography)

EMDB-25466, PDB-7sw8:
MicroED structure of proteinase K from a 150 nm thick lamella measured at 300 kV
Method: EM (electron crystallography)

EMDB-25467, PDB-7sw9:
MicroED structure of proteinase K from a 170 nm thick lamella measured at 300 kV
Method: EM (electron crystallography)

EMDB-25468, PDB-7swa:
MicroED structure of proteinase K from a 320 nm thick lamella measured at 300 kV
Method: EM (electron crystallography)

EMDB-25469, PDB-7swb:
MicroED structure of proteinase K from a 360 nm thick lamella measured at 300 kV
Method: EM (electron crystallography)

EMDB-25470, PDB-7swc:
MicroED structure of proteinase K from a 550 nm thick lamella measured at 300 kV
Method: EM (electron crystallography)

Chemicals

ChemComp-HOH:
WATER / Water

Source
  • parengyodontium album (fungus)
KeywordsHYDROLASE / Serine protease

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