(My apologies to Ken Williams!)
Protein Structures Without Crystals:
Single-particle Electron Cryo-Microscopy
Fred J. Sigworth
Department of Cellular and Molecular Physiology
Department of Biomedical Engineering
Yale University
Cryo-EM protein structure determination: 2D crystals
electrons
C
Electron crystallography
Cryo-EM protein structure determination: single particles
electrons
C
Single-particle cryo-EM
The Cryo-EM Specimen
EM grid
3 mm
Grid square
Hole in carbon film
60 µm
1 µm
A Cryo-EM Specimen
500 nm
Ice spanning a hole
Yale’s Tecnai F20 electron cryo-microscope.
It has a field-emission source for high
resolution imaging of phase-contrast
specimens.
A single-particle micrograph
From our $900k microscope:
Solubilized IP3 receptors.
Processing the single-particle images
1. Original micrograph
2. Normalized and centered particle images
Boxed Particles
3. Class Averages
1000 Å
Class averages from IP3R particle images
Here are compared
projections from a 3D
model (left columns) and
the experimental class
averages (right columns)
[ alfa,beta ] = [
[
[
[
[
0.0 ,
33.60 ,
57.60 ,
67.20 ,
86.40 ,
0.0 ] , [
20.00 ] , [
38.57 ] , [
54.00 ] , [
33.75 ] , [
4.80 , 0.0 ] , [ 33.60 , 10.00 ] ,
48.00 , 7.50 ] , [ 48.00 , 15.00 ] ,
57.60 , 45.00 ] , [ 67.20 , 48.00 ] ,
76.80 , 45.00 ] , [ 76.80 , 50.62 ] ,
86.40 , 39.37 ]
The Ryanodine Receptor, and our first IP3 Receptor structure.
Both of these intracellular Ca2+-release channels have been studied only by EM.
The computational magic:
deducing the 3D structure from projections
Example of 3D reconstruction
Fourier transforms
The Projection Theorem, well known to medical imaging, allows
the reconstruction of 3D volumes from projections.
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Projection images
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The Fourier transforms of images are inserted as planes into a
3D volume. The inverse transform then yields the 3D structure.
Ca-ATPase map thanks to D. L. Stokes
Example of 3D reconstruction
Projection images
Fourier transforms
A better reconstruction from 9 projections.
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Common lines method for orienting projections
Every pair of 2D projections shares a common 1D projection (a “common line”).
The common lines show the relative directions of the projections.
How many particle images are needed?
N
Resolution
2k
20 Å
70k
10 Å
1M
4Å ?
Why are so many images required?
Log Noise
Log Signal
The signal-to-noise ratio of a single image is far below unity.
Many images must be averaged to get SNR>1, especially at high resolution.
Resolution
Rosenthal and Henderson, J. Mol. Biol. 2003
At present, the record for resolution is about 6 Å.
The Gro-EL single-particle study by
Ludtke et al. (Structure 2004) used
40,000 particles.
Because GroEL has symmetry (14
subunits per particle) this is equivalent to
280,000 asymmetric units.
Resolution is estimated by the Fourier
Shell Correlation function.
GroEL from single particles
The 6 Å cryo-EM density map is
detailed enough to allow the
identification of alpha helices.
How to acquire all these images?
The Leginon software
system developed by
Carragher, Potter and
colleagues, performs
automatic EM data
acquisition.
We are setting up this
system on our microscopes.
The Leginon system
Leginon acquires
images and identifies
“interesting” regions in
a hierarchical fashion
at magnifications from
100x to 100,000x
Some technical challenges in single-particle cryo-EM
1.
Particle selection from micrographs
2.
Imaging membrane proteins in membranes
3.
Improving the phase contrast
1. Particle selection
The challenge is to find thousands of particles automatically. Here’s a snapshot from the
computer program we entered into the Scripps particle-selection bakeoff.
Shirley Wang
2. Imaging membrane proteins in membranes
The spherical reconstruction strategy allows membrane proteins to be imaged
And two orientation angles determined
Simulated images of Ca-ATPase in vesicles
Fitting and subtracting the membrane image
A “scalable” membrane
model is fitted to an EM
image and subtracted. The
resulting image can be
processed for single-particle
reconstruction.
3. The prospect of better phase-contrast imaging
The Zernike phase plate is well known in light
microscopy.
Boersch proposed an electrostatic phase plate in
the 1940s.
Electrons passing through the center of the
device experience ~40 V•µm of potential, shifting
their phase by π/2.
The trick will be to fabricate a 1 µm or smaller
device, suspended in the middle of a 30 µm
aperture disc.
The contrast of cryo-EM images could be increased.
Danev and Nagayama (2001)
have demonstrated a
microfabricated phase-plate
which gives a much better
contrast-transfer function.
Cryo-EM Technology
There’s lots to do. Why not work with us on cryo-EM technology?
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Liguo Wang
Chris Cantener
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
David Chester
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Quic kTime™ and a
TIFF (Unc ompres sed) dec ompres sor
are needed to see this pic ture.
Puey Ounjai
Sigworth Lab
Collaborators
K Channels
Youshan Yang
Teresa Giráldez
Chris Cantener
Shumin Bian
Yangyang Yan
Planar Patch Clamp
Electrodes
Kate Klemic
Xiaohui Li
Farah Laiwalla
IP3 Receptors
David Chester
Bill Grenawitzke
EM Technology
Liguo Wang
David Chester
Puey Ounjai
Paul Sawroop
Barbara Ehrlich
Ed Moczydlowski
Eugenio Culurciello
Mark Reed
Jim Klemic
250 Å