LIFE SCIENCE TECHNOLOGIES
SCIENCE 1393microscopyProduced by the Science/AAAS Custom Publishing OfficeInstead of analyzing images of extracted proteins or complexes as SPA does,
cryo-ET collects data on one cell, getting multiple views by tilting it on a grid.
Averaging the images in a process similar to SPA results in 3D reconstructions.
TEM requires samples thinner than most cells, so researchers like Villa pair
cryo-ET with focused ion-beam (FIB) micromachining. Villa credits Mike Marko
at the New York State Department of Health for showing that this typical
materials science method is feasible for vitrified samples. As a postdoc in
Wolfgang Baumeister’s lab at the Max Planck Institute of Biochemistry in
Munich, Germany, Villa helped advance the technique.
Cryo-FIB uses a special cryo-EM instrument that aims a beam of large ions,
such as gallium, across a vitrified sample to plane it down. “You blast it on the
top and bottom until you have a window sustained by the sides of the cell that
is thin enough for cryo-ET,” Villa says. If a cell’s tomography shows multiple
copies of a protein or complex, subtomogram averaging of individual images
of that molecule can reconstruct its 3D structure.
Villa’s lab added correlative light and electron microscopy (CLEM) to cryo-ET
to determine the 14-Å structure of LRRK2. The researchers tagged the protein,
which is involved in Parkinson’s disease, to locate it in cells using CLEM. Then
they used FIB to mill down the cells for cryo-ET to obtain in situ 3D structures
of LRRK2, including its close association with microtubules ( 9 ). LRRK2 can’t be
crystallized, “but with cryo-ET and subtomogram averaging, we solved the
structure while it was still in the cell,” Villa says.
According to Villa, improvements ahead for cryo-ET include developing
specialized TEM grids for growing cells before inducing a change, such as
stimulating neurons or exposing human cells to medicines. Researchers usinglight microscopy could choose when to vitrify the sample for cryo-ET, Villa says,
“to see at high resolution what happened at the point you did something to
the cell.”
Sample preparation for cryo-ET is low throughput, but a variation of CLEM
with multiplexed fluorescent markers, developed by John Briggs’ group at
the European Molecular Biology Laboratory in Germany, could more quickly
identify cells for cryo-ET. Cryo-ET will allow observation of molecules in their
native environment and in whole tissues, such as molecular views of “the
connectome” of neuron-to-neuron interactions, Nissen says. The Thermo
Fisher second-generation FIB instrument, notes Reyntjens, has cryo-liftout
capability for manipulating miniscule samples cut from vitrified tissue thinned
to 100 nm–150 nm and transferred to a cryo-EM instrument for tomography.
NIH will soon launch national cryo-ET centers. The current cryo-EM centers,
Carragher explains, collect tomograms if users have samples ready for cryo-ET.
The national centers will provide access to equipment, plus assistance with
tricky cryo-ET specimen preparation.
In addition to developments like CLEM, structural analysis that combines
cryo-EM with data from multiple sources is on the rise. “Increasingly, people
use crystallography, NMR, CLEM, mass spec—everything out there—to get an
answer,” Carragher says. “But if we want these tools in everyone’s toolkit, they
need to be more accessible.”New ambitions
Along with solving the access problem, Nissen observes, the field should
shift its perspective from focusing only on structures to “asking what the
structure is doing in the cell in its native state. Getting label-free, time-
resolved structures in natural contexts is the ultimate goal and also a new level
of ambition to instill in students and postdocs.”
Nissen and others predict increasing industry use of cryo-EM for developing
antibody therapeutics, small molecule drugs, and diagnostics. “We should also
work with the medical community on unmet diagnostic needs,” he says, “where
histology doesn’t show good differences between disease and healthy tissue.
We might find molecular differences in tissues [by also] using cryo-ET.”
van Heel, who helped develop cryo-EM and has watched its use grow, says
about working in the field, “It’s challenging at the moment, but it’s a great
time to be alive. There’s no time for vacation.”References- Y. Cheng, Science 361 , 876–880 (2018), doi: 10.1126/science.aat4346.
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Chris Tachibana is a freelance writer who specializes in life sciences.Aarhus University
international.au.dk
Brazilian Nanotechnology National
Laboratory
lnnano.cnpem.br
Gonen Lab, University of California,
Los Angeles
cryoem.ucla.edu
NanoImaging Services
http://www.nanoimagingservices.com
New York Structural Biology Center
nysbc.orgFeatured participantsOregon Health & Science
University
http://www.ohsu.edu
Structura Biotechnology
structura.bio
Thermo Fisher Scientific
w w w.T hermoFisher.com
Villa Lab, University of California,
San Diego
villalab.ucsd.eduPHOTO: COURTESY OF OREGON HE/LTH & SCIENCE UNIVERSITY/KRISTYN/ WENTZ-GR/FF
Craig Yoshioka, codirector of the NIH cryo-EM center at Oregon Health & Science University,
stands in front of one of his facility’s cryo-EM instruments.