architecture of the photosynthetic apparatus by electron microscopy
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Architecture of the photosynthetic apparatus by electron microscopy. Egbert Boekema Leiden March 2009. Dear keynote speakers in our Solar Biofuels of Microorganisms Workshop - PowerPoint PPT PresentationTRANSCRIPT
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Architecture of the photosynthetic
apparatus
by electron microscopy
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 <http://www.lorentzcenter.nl/lc/web/2009/333/info.php3?wsid=333>,
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
Photosystem I trimer+ 18 antenna proteins
“single particle averaging”
antenna protein =IsiA, the iron stressinduced protein Aof 37 kDa
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main steps in single particle averaging
• EM + selection of particle projections• alignment of randomly oriented projections
rotational + translational shifts• sorting of projections
statistical analysis + classification• calculation of two-dimensional projection maps
summing of projections into “classes”• calculation of 3D structures
• EM + selection of particle projections• alignment of randomly oriented projections
rotational + translational shifts• sorting of projections
statistical analysis + classification• calculation of two-dimensional projection maps
summing of projections into “classes”• calculation of 3D structures
tilted classsymmetrical
class tilted class
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Resolution in single particle cryo-3D reconstructions
object mass numberprojections
resolution(Å)
symmetry
70 s ribosome
worm hemoglobin 4000 kDa
3000 kDa none
12-fold
4,00020,00075,000
1,000
251710
13
GroEL 823 kDa 14-fold 10,000 840,000 6
Ca release channel 2300 kDa 4-fold 22,000 14
290 kDa 2-fold 36,000 7.5Transferrin receptor -transferrin complex
protein 6 rotavirus >20-fold 8,400 4>5000 kDa
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First protein at atomic resolution
viral protein 6 in rotavirus DLP: 8,400 particles8,400 x 60 x 13 = 6.6 million copies
Zhang et al. PNAS 2008, 105, 1867
Cryo-EM imageAssignment of amino acidside chains in the 3D map
Electron density map plusamino acid side chain fit(blue wires)
lower resolution presentation of
virus reconstruction
Cryo-EM picture showingVirus particles in a thin layerof ice of a holey carbon film
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A test object: worm hemoglobinA test object: worm hemoglobin
100 Å100 Å
12 x 12 proteins12 x 12 proteins
18 linker proteins18 linker proteins
18 linker proteins18 linker proteins
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Most complicated step in single particle averaging
sorting of projectionsstatistical analysis + classificationsorting of projectionsstatistical analysis + classification
tilted classsymmetrical
class tilted class
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Gallery of aligned top- and side views Gallery of aligned top- and side views
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Classification map after statistical analysis
Factor 1Factor 1
Factor 2Factor 2
Each dot is a particle in side-view position close in space = high similarity
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Classification of aligned Classification of aligned
side-view projectionsside-view projections
HH
BB CC DD
EE FF GG
AA
II
partition of data set into 9 classespartition of data set into 9 classes
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Position of classes in the classification Position of classes in the classification mapmap
EE
AA
HH
BB
CC
DD
FF
GG
II
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Relationship Relationship betweenbetween side-views side-views andand top viewstop views
Support filmSupport film
Support filmSupport film
beambeam
predominantpredominantposition 1position 1
predominantpredominantposition 2position 2
““broad type”broad type”side viewside view
““narrow type”narrow type”side viewside view
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Sinograms of individual hemoglobin classes Sinograms of individual hemoglobin classes to find to find searching common lines
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Worm Hemoglobin 3D ModelWorm Hemoglobin 3D Model
EM X-ray
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19881988
Photographic emulsionPhotographic emulsion5000 particles5000 particles1 minute / particle1 minute / particle
20092009
CCD cameras (200,000 CCD cameras (200,000 €€))50,000 particles50,000 particles1000 particles / minute1000 particles / minute
> 2010> 2010
Handcraft Semi-automation remote-control
Atomic resolutionAtomic resolutionelectron counters electron counters
(800,000 (800,000 €€))500,000 particles500,000 particles10000 particles / minute10000 particles / minute
sum of 1024 particles
11 11 Å resolutionÅ resolutionin negative stainin negative stain
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Seeing is believing
The skull from Dali
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Seeing is believing
EM(18 Å resolution)
X-ray
Complex III(Cytochrome reductase)
The skull from Dali
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Example of combining EM and X-ray Example of combining EM and X-ray diffractiondiffraction
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
PSIIPSIIPSIPSIATPaseATPase CytbCytb66ff
PhycobilisomePhycobilisome
NDH-1NDH-1
Cyanobacteria do not have a membrane-bound antenna with LHCH2Cyanobacteria do not have a membrane-bound antenna with LHCH2
Rows of PSII are a scaffold for the phycobilisomes but nobody knows howRows of PSII are a scaffold for the phycobilisomes but nobody knows how
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PBS components known at high resolutionPBS components known at high resolution
Phycobilisomes are floppy: Structure work on truncated PBSsPhycobilisomes are floppy: Structure work on truncated PBSs
Need for solving interaction with PSII-PSI, FNR, quenching proteinsNeed for solving interaction with PSII-PSI, FNR, quenching proteins
Phycobilisome (PBS)
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Single particle analysis of PBSsSingle particle analysis of PBSs
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Single particle electron microscopydigitonin-solubilized cyanobacterial membranes
Photosystem 2Photosystem 2
Complex IComplex IATPaseATPase
Photosystem 1Photosystem 1
50 nm50 nm
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Selected gallery of projection maps from 15,000 projections
733733 351351 304304
512512 512512 218218
312312
12301230
~300~300 5050 291291 77
Performed on Synechocystis 6803 / Thermocynechococcus elongatusPerformed on Synechocystis 6803 / Thermocynechococcus elongatus
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Seeing is believing Some ’’’assignments’’’
Glutamine synthaseGlutamine synthase
PhycobilisomePhycobilisome
fragmentfragment
ATP synthaseATP synthaseT-shaped T-shaped particleparticle GroEl-GroESGroEl-GroES
from the PDB sitefrom the PDB site““molecule of themolecule of themonth displays”month displays”
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Small Photosystem II arrays in solubilized Small Photosystem II arrays in solubilized membranes from Synechocystis 6803membranes from Synechocystis 6803
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Analysis of Photosystem II arrays and double Analysis of Photosystem II arrays and double dimersdimers
Phycobilisome modelPhycobilisome model
16.7 nm16.7 nm
12.5 nm12.5 nm
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Analysis of Photosystem II double dimersAnalysis of Photosystem II double dimers
Double dimer modelDouble dimer model
Is there a specific subunit involved in double dimer formation?Is there a specific subunit involved in double dimer formation?
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Models for the photosynthetic membraneModels for the photosynthetic membrane