well as the concurrent use of cryogenic techniques to minimize
radiation damage [1]. We demonstrate the implementation of this
technique on GSNQNNF, a routinely studied heptapeptide segment of the prion protein [12], which forms needle-like crystals
several microns in length but no more than a few hundred nanometers thick and wide.
As with the interrogation of larger macromolecular structures
by MicroED, data collection from frozen hydrated crystals proceeds by measuring diffraction from a selected area of an illuminated crystal while that crystal is rotated unidirectionally in the
electron beam [1, 13]. Diffraction movies recorded during this
process are then converted into formats accessible to conventional
crystallographic processing programs, which reduce MicroED data
to intensities [13] used for ab initio phasing by direct methods.
Solutions obtained by this approach contain collections of atoms
which display correlations with the measured data. These in turn
can generate electrostatic potential maps that inform the placement
of residues along a polypeptide chain. Fourier difference density in
these structures may reveal the positions of riding hydrogens, a feat
difficult to accomplish via conventional X-ray crystallographic
methods [5, 6, 14].
The procedures outlined in this chapter assume a certain degree
of practical and theoretical knowledge related to operation of
transmission electron microscopes, cryoEM, crystallography, and
diffraction theory. For a primer on these subjects the reader is
encouraged to read additional chapters in this book, or one of
several recent reviews or books [1, 15–18].
2 Materials
2.1 SamplePreparation Tools and
Consumables
1. 200–400 mesh grids, covered with continuous or perforated
carbon.
2. Ultra-fine grade reverse self-closing tweezers.
3. Vitrobot fine-tip tweezers.
4. Whatman No. 1 filter paper for specimen blotting and standard
Vitrobot filter paper.
5. Glass petri dish with lid for grid storage and transport.
6. 2, 10, 20 μL micropipettors for sample manipulation and
application.
7. Crystal slurries prepared in batch, or in hanging or sitting drops
via vapor diffusion or other crystal growth and storage system.
8. Cryogen-resistant grid storage boxes with screw-top tool.
9. Personal protective equipment: several procedures require use
of safety goggles, gloves, and cryogen protection including
cryogen gloves and face shields.
330
Chih-Te Zee et al.
radiation damage [1]. We demonstrate the implementation of this
technique on GSNQNNF, a routinely studied heptapeptide segment of the prion protein [12], which forms needle-like crystals
several microns in length but no more than a few hundred nanometers thick and wide.
As with the interrogation of larger macromolecular structures
by MicroED, data collection from frozen hydrated crystals proceeds by measuring diffraction from a selected area of an illuminated crystal while that crystal is rotated unidirectionally in the
electron beam [1, 13]. Diffraction movies recorded during this
process are then converted into formats accessible to conventional
crystallographic processing programs, which reduce MicroED data
to intensities [13] used for ab initio phasing by direct methods.
Solutions obtained by this approach contain collections of atoms
which display correlations with the measured data. These in turn
can generate electrostatic potential maps that inform the placement
of residues along a polypeptide chain. Fourier difference density in
these structures may reveal the positions of riding hydrogens, a feat
difficult to accomplish via conventional X-ray crystallographic
methods [5, 6, 14].
The procedures outlined in this chapter assume a certain degree
of practical and theoretical knowledge related to operation of
transmission electron microscopes, cryoEM, crystallography, and
diffraction theory. For a primer on these subjects the reader is
encouraged to read additional chapters in this book, or one of
several recent reviews or books [1, 15–18].
2 Materials
2.1 SamplePreparation Tools and
Consumables
1. 200–400 mesh grids, covered with continuous or perforated
carbon.
2. Ultra-fine grade reverse self-closing tweezers.
3. Vitrobot fine-tip tweezers.
4. Whatman No. 1 filter paper for specimen blotting and standard
Vitrobot filter paper.
5. Glass petri dish with lid for grid storage and transport.
6. 2, 10, 20 μL micropipettors for sample manipulation and
application.
7. Crystal slurries prepared in batch, or in hanging or sitting drops
via vapor diffusion or other crystal growth and storage system.
8. Cryogen-resistant grid storage boxes with screw-top tool.
9. Personal protective equipment: several procedures require use
of safety goggles, gloves, and cryogen protection including
cryogen gloves and face shields.
330
Chih-Te Zee et al.