Architecture of the photosynthetic

apparatus

by electron microscopy

Architecture of the photosynthetic

apparatus

by electron microscopy

Egbert Boekema

Leiden March 2009

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

Unfiltered image of a copper phtalocyanin crystal

Electron microscopy is possible at atomic resolution

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

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

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

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

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

Most complicated step in single particle averaging

sorting of projectionsstatistical analysis + classificationsorting of projectionsstatistical analysis + classification

tilted classsymmetrical

class tilted class

Gallery of aligned top- and side views Gallery of aligned top- and side views

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

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

Position of classes in the classification Position of classes in the classification mapmap

EE

AA

HH

BB

CC

DD

FF

GG

II

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

Sinograms of individual hemoglobin classes Sinograms of individual hemoglobin classes to find to find searching common lines

Worm Hemoglobin 3D ModelWorm Hemoglobin 3D Model

EM X-ray

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

Seeing is believing

The skull from Dali

Seeing is believing

EM(18 Å resolution)

X-ray

Complex III(Cytochrome reductase)

The skull from Dali

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

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

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)

Single particle analysis of PBSsSingle particle analysis of PBSs

Single particle electron microscopydigitonin-solubilized cyanobacterial membranes

Photosystem 2Photosystem 2

Complex IComplex IATPaseATPase

Photosystem 1Photosystem 1

50 nm50 nm

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

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”

Small Photosystem II arrays in solubilized Small Photosystem II arrays in solubilized membranes from Synechocystis 6803membranes from Synechocystis 6803

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

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?

Models for the photosynthetic membraneModels for the photosynthetic membrane