Architecture of the photosynthetic

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Architecture of the photosynthetic apparatus
by electron microscopy
Egbert Boekema Leiden March 2009
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Dear keynote speakers in our Solar Biofuels of
Microorganisms Workshop lthttp//www.lorentzcenter
.nl/lc/web/2009/333/info.php3?wsid333gt,The
workshop is embedded in the Leiden University
Honours programme, and there will be 20 of our
best bachelor students participating. We
havecomfortable slots for the talks and the
discussion, and with this email I would like to
ask you not to hesitate to include an educational
dimension inyour lecture, it will be
appreciated, both by our students and by the
participant out side your own field in this
multidisciplinary workshop.Thanking you for
your efforts, and looking forward to seeing you
soon in Leiden.Kind regards,-on behalf of the
organizers-Corrie Kuster
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Unfiltered image of a copper phtalocyanin crystal
Electron microscopy is possible at atomic
resolution
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removal of noise by averaging of many images
single particle averaging
antenna protein IsiA, the iron
stress induced protein A of 37 kDa
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main steps in single particle averaging

• EM selection of particle projections
• alignment of randomly oriented projections rotati
  onal translational shifts
• sorting of projections
• statistical analysis classification
• calculation of two-dimensional projection maps
• summing of projections into classes
• calculation of 3D structures

symmetrical class
tilted class
tilted class
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Resolution in single particle cryo-3D
reconstructions
object
mass
number projections
resolution (Å)
symmetry
25 17 10
4,000 20,000 75,000
70 s ribosome
3000 kDa
none
worm hemoglobin
12-fold
1,000
13
4000 kDa
Transferrin receptor - transferrin complex
290 kDa
2-fold
36,000
7.5
14
2300 kDa
4-fold
22,000
Ca release channel
823 kDa
14-fold
10,000
GroEL
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40,000
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protein 6 rotavirus
gt5000 kDa
gt20-fold
8,400
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First protein at atomic resolution
viral protein 6 in rotavirus DLP 8,400
particles 8,400 x 60 x 13 6.6 million
copies Zhang et al. PNAS 2008, 105, 1867
Cryo-EM image
Electron density map plus amino acid side chain
fit (blue wires)
lower resolution presentation of virus
reconstruction
Assignment of amino acid side chains in the 3D map
Cryo-EM picture showing Virus particles in a thin
layer of ice of a holey carbon film
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A test object worm hemoglobin
12 x 12 proteins
18 linker proteins
18 linker proteins
100 Å
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Most complicated step in single particle averaging
sorting of projections statistical analysis
classification
symmetrical class
tilted class
tilted class
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Gallery of aligned top- and side views
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Classification map after statistical analysis
Factor 2
Factor 1
Each dot is a particle in side-view position
close in space high similarity
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Classification of aligned side-view projections
C
D
B
A
G
H
E
I
F
partition of data set into 9 classes
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Position of classes in the classification map
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Relationship between side-views and top views
predominant position 1
broad type side view
Support film
narrow type side view
predominant position 2
Support film
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Sinograms of individual hemoglobin classes to
find searching common lines
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Worm Hemoglobin 3D Model
EM
X-ray
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1988
2009
11 Å resolution in negative stain
gt 2010
Atomic resolution electron counters (800,000
) 500,000 particles 10000 particles / minute
CCD cameras (200,000 ) 50,000 particles 1000
particles / minute
Photographic emulsion 5000 particles 1 minute /
particle
Semi-automation
remote-control
Handcraft
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Seeing is believing
The skull from Dali
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Seeing is believing
The skull from Dali
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Example of combining EM and X-ray diffraction
Cytochrome reductase and cytochrome oxidase
supercomplex (Heinemeyer et al. 2007 J. Biol.
Chem. 282, 12240
maps of the supercomplex and a fragment (left)
show enough fine structure to dock the complex
III and IV crystal structures accurately into the
EM density maps
Conclusion from 15 Å EM data X-ray structures
we get a pseudo-atomic model, which has enough
resolution to predict interaction of alpha
helices of different subunits
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Scheme of the cyanobacterial membrane
Phycobilisome
ATPase
PSII
Cytb6f
PSI
NDH-1
Cyanobacteria do not have a membrane-bound
antenna with LHCH2
Rows of PSII are a scaffold for the
phycobilisomes but nobody knows how
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Phycobilisome (PBS)
PBS components known at high resolution Phycobili
somes are floppy Structure work on truncated
PBSs Need for solving interaction with PSII-PSI,
FNR, quenching proteins
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Single particle analysis of PBSs
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Single particle electron microscopy digitonin-solu
bilized cyanobacterial membranes
50 nm
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Selected gallery of projection maps from 15,000
projections
Performed on Synechocystis 6803 /
Thermocynechococcus elongatus
733
351
304
312
512
512
218
1230
50
7
300
291
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Seeing is believing Some assignments
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Small Photosystem II arrays in solubilized
membranes from Synechocystis 6803
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Analysis of Photosystem II arrays and double
dimers
16.7 nm
12.5 nm
Phycobilisome model
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Analysis of Photosystem II double dimers
Double dimer model
Is there a specific subunit involved in double
dimer formation?
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Models for the photosynthetic membrane