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Electron Crystallography
Henning Stahlberg
http://C-CINA.org
3D model(small
proteins)
3D modelResolution:
12 Å
3D modelResolution: x,y: 2.5 Åz: 3.0 Å
2D surface Resolution:
x,y: 5 Åz: 1 Å
3D modelResolution:
1.5 Å
Nuclear Magn.Resonance
Atomic ForceMicroscopy
X-Ray Diffraction
Single Particles
2DCrystallization
3D Crystallization
Purified (Membrane) Protein
Electron Microscopy
3D modelResolution: x,y: 2.5 Åz: 3.0 Å
2D surface Resolution:
x,y: 5 Åz: 1 Å
3D model(small
proteins)
3D modelResolution:
12 Å
3D modelResolution:
2 Å
Nuclear Magn.Resonance
Atomic ForceMicroscopy
X-Ray Diffraction
Single Particles
3D Crystallization
Purified (Membrane) Protein
Electron Microscopy
2DCrystallization
Single Particle TEM
Expression:His-glpF
in E. coli,
solubilizedwith OG,
purified onNi-Agarose,
neg. stainedsingle particles,
TEM image
GlpF
3D modelResolution: x,y: 2.5 Åz: 3.0 Å
2D surface Resolution:
x,y: 5 Åz: 1 Å
3D model(small
proteins)
3D modelResolution:
12 Å
3D modelResolution:
2 Å
Nuclear Magn.Resonance
Atomic ForceMicroscopy
X-Ray Diffraction
Single Particles
3D Crystallization
Purified Membrane Protein
Electron Microscopy
2DCrystallization
Imaging
Purification
Sample Prep
Image Processing
Model Building
2D Crystallization
Imaging
Bacterial
Yeast
Insect
Mammalian
Cell-Free
Expression
Sample Prep
Image Processing
Model Building
2D Crystallization
Expression
Imaging
lipiddouble layer membrane
protein
detergentmolecule
solubilized membrane protein
detergentmicelles
Solubilization: FOS-12…14, DHPC, DDM
Purification: DMReconstitution: OTG, OG
Detergents
Purification
The Transmission Electron Microscope
-200’000 Volt !
Sample
Viewing Screen
Vacuum !
Negative Stain EM Grid Preparation-e
Proteins
Negative Stain
Carbon-Film
BF-TEM image of negatively stained MloK1 membrane protein
50 nm
Sample Prep
Image Processing
Model Building
2D Crystallization
Expression
Imaging
Chiu et al., Structure 2007
Purification
BF-TEM image of negatively stained MloK1 membrane protein
50 nm
52 Å
Sample Prep
Image Processing
Model Building
2D Crystallization
Expression
Imaging
Chiu et al., Structure 2007
Purification
Purification
Sample Prep
Image Processing
Model Building
Expression
Imaging
2D Crystallization
Jap et al., Ultramicroscopy (1992)
SampleDialysis
Membrane
Detergent removal by dialysis
Detergent capture by adding cyclodextrin
Purification
Sample Prep
Image Processing
Model Building
Expression
Imaging
2D Crystallization
Iacovache et al., J. Struct. Biol. (2010)
(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
!"(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
!#(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
!$(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
!%(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
!&(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
'((c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
'!(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
(c) Slide from presentation given by Tom Walz, Harvard, on the Electron Crystallography Workshop at UC Davis, 2008
Cryo-EM Grid Preparation-e
Proteins
Vitrified Water
Carbon-Film
2D Crystals of Membrane Proteins
2 µm
Imaging
Electron Diffraction
Goals:
• User-friendly• Fast to install (Linux, OSX)
• Compatible (MRC, CCP4, SPIDER, Chimera, EMDEP, PDB...)
• Easy to learn and to get started• Transparent• Allows manual fine-tuning of every step• Flexible (easy to adapt to your own programs)
• Optionally fully automated• Include new algorithms
Image Processing
“Magic”ImageProcessing
MRC programsRichard Henderson et al.
Available from Jude Smith, MRC, Cambridge, UK
BryantGipson
MarcelArheit
Extract the information from the noiseFFTNoisy
image
Filtered FFT
Filtered image
Fourier Filtering
Image
FFT
Fourier Filtering
Image
FFT
Lattice Indexed(in FFT)
Fourier Filtering
Image
FFT
Lattice Indexed(in FFT)
Masked(in FFT)
Filtered Image
Fourier Filtering
Image
FFT
Lattice Indexed(in FFT)
Masked(in FFT)
Reconstruction (2D)
Reconstruction (3D)
Filtered Image
CTF world
Basic Unbending Algorithm
FFT
FFT MaskedFFT
mask= 1px
masktran
MaskedFFT
20px
PerfectFFT
UnbentImage
RawImage
Reference
FFT of ReferenceCC-Map
quadserchccunbendProfile
CCP4ctfapply
Amps&
Phases origtilt
Amps&
Phases
Collection of Amp&Phs
Amps&
PhasesAmps
&Phases
Amps&
PhasesAmps
&Phases
Amps&
PhasesAmps
&Phases
Amps&
Phases
Amps&
Phases
mmbox
maketran
(ApplyDefocus)
More Complete Unbending Algorithm
RawFFT
MaskedFFT
(24px)
UnbentImage
(III)
FFT of UnbentImage
(III)
Amps&
Phases(APH file)
CTF-corr.Amps &Phases
(APH file) Collectionof
APH files
MergedAmps &Phases
(APH file)
From other images
RawImage
MaskedFFT
(1 px)Reference
FFTCropped
Reference(400px)
Cross-Cor. Map
1
DistortionVectorField
1MaskedFFT
(20px)
MaskedFFT
(2 px)Reference
FFT
CroppedReference
(300px)Distortion
VectorField
2MaskedFFT
(22px)
UnbentImage
(I)
FFT of UnbentImage
(I)
MaskedFFT
(2 px)Reference
FFT
CroppedReference
(300px)Distortion
VectorField
3
UnbentImage
(II)
FFT of UnbentImage
(II)
Cross-Cor. Map
2
Cross-Cor. Map
3
UNBEND I
UNBEND II (1st iteration)
UNBEND II (2nd iteration)
Cryo-EM
Continous C-film
-180°C
Before!Image Processing
Cryo-EM
Continous C-film
-180°C
Before!! After Image Processing
Cryo-EM
Continous C-film
-180°C
Before!! After Image Processing
Real Space Fourier Space
x
y
z
The Third Dimension
Real Space Fourier Space
Diffraction Spots in 2D
The Third Dimension
Real Space Fourier Space
Diffraction Spots in 2D
Lattice Lines in 3D
x
y
z
Complex Values for Amplitude and Phase
in Z-direction
The Lattice Line
Fourier Space
0
Resolution in Z-direction0
The Radon projection theorem for 3D reconstructions
Fourier-Space Method
The Missing Cone in Fourier SpaceFourier Space
Diffraction Spots in 2DLattice Lines in 3D
Real Space
3D Reconstruction Cryo EMGlpF
A first 3D Map of a Waterchannel
AQP1
3D cryo TEM3.8 Å resolution
Mitsuoka et al. J. Struct. Biol. (1999)
Atomic model
3D cryo EM, 3.8 Å resolution, Murata et al. Nature 2000
Interpretation
3D cryo EM, 3.8 Å resolution, Murata et al. Nature 2000
Conclusions• Water channel is
amphipathic
• H20 traverses in a 1D string
• 2 Asn dissect string, positive residues and helix dipoles: all inhibit permeation of H3O+
Grotthus “hop (I) and turn (II)” proton conduction mechanism
De Grotthuss, C.J.T. (1806). Mémoire sur la décomposition de l’eau et des corps qu’elle tient en dissolution a l’aide de l’électricité galvanique. Ann. Chim. 58, 54-74
Molecular dynamics
Bert de Groot & Helmut Grubmüller, Science (2001)300 K, 4 AQP1, 271 lipid, 25’891 H2O10 ns molecular dynamics simulation
The Nobel Prize in Chemistry 2003
"for discoveries concerning channels in cell membranes"
Peter Agre
Johns Hopkins University School of Medicine Baltimore, MD, USA
b. 1949
Roderick MacKinnon
Rockefeller University, Howard Hughes Medical Institute
New York, NY, USA b. 1956
"for the discovery of water channels"
"for structural and mechanistic studies of ion
channels"