1. Architecture of the photosynthetic apparatus by electron microscopy Architecture of the photosynthetic apparatus by electron microscopy Egbert Boekema Leiden March 2009

2. Dear keynote speakers in our Solar Biofuels of Microorganisms Workshop, The workshop is embedded in the Leiden University Honours programme,http://www.lorentzcenter.nl/lc/web/2009/333/info.php3?wsid=333 and there will be 20 of our best bachelor students participating. We have comfortable 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 in your 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

3. Unfiltered image of a copper phtalocyanin crystal Electron microscopy is possible at atomic resolution

4. removal of noise by averaging of many images Photosystem I trimer + 18 antenna proteins “single particle averaging” antenna protein = IsiA, the iron stress induced protein A of 37 kDa

5. 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 class symmetrical class tilted class

6. Resolution in single particle cryo-3D reconstructions object mass number projections resolution (Å) symmetry 70 s ribosome worm hemoglobin 4000 kDa 3000 kDa none 12-fold 4,000 20,000 75,000 1,000 25 17 10 13 GroEL 823 kDa14-fold10,000 8 40,000 6 Ca release channel 2300 kDa4-fold22,000 14 290 kDa2-fold36,000 7.5 Transferrin receptor - transferrin complex protein 6 rotavirus >20-fold8,400 4 >5000 kDa

7. 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 Assignment of amino acid side chains in the 3D map Electron density map plus amino acid side chain fit (blue wires) lower resolution presentation of virus reconstruction Cryo-EM picture showing Virus particles in a thin layer of ice of a holey carbon film

8. A test object: worm hemoglobin 100 Å 12 x 12 proteins 18 linker proteins

9. Most complicated step in single particle averaging sorting of projections statistical analysis + classification sorting of projections statistical analysis + classification tilted class symmetrical class tilted class

10. Gallery of aligned top- and side views

11. Classification map after statistical analysis Factor 1 Factor 2 Each dot is a particle in side-view position close in space = high similarity

12. Classification of aligned side-view projections H B C D E F G A I partition of data set into 9 classes

13. Position of classes in the classification map EA H B CDF GI

14. Relationship between side-views and top views Support film beampredominant position 1 predominant position 2 “broad type” side view “narrow type” side view

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

16. Worm Hemoglobin 3D Model EMX-ray

17. 1988 Photographic emulsion 5000 particles 1 minute / particle 2009 CCD cameras (200,000 €) 50,000 particles 1000 particles / minute > 2010 Handcraft Semi-automation remote-control Atomic resolution electron counters (800,000 €) 500,000 particles 10000 particles / minute sum of 1024 particles 11 Å resolution in negative stain

18. Seeing is believing The skull from Dali

19. Seeing is believing EM (18 Å resolution) X-ray Complex III (Cytochrome reductase) The skull from Dali

20. 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

21. Scheme of the cyanobacterial membrane PSII PSI ATPase Cytb 6 f Phycobilisome 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

22. PBS components known at high resolution Phycobilisomes are floppy: Structure work on truncated PBSs Need for solving interaction with PSII-PSI, FNR, quenching proteins Phycobilisome (PBS)

23. Single particle analysis of PBSs

24. Single particle electron microscopy digitonin-solubilized cyanobacterial membranes Photosystem 2 Complex I ATPase Photosystem 1 50 nm

25. Selected gallery of projection maps from 15,000 projections 733351304 512 512 218 312 1230 ~300 50 291 7 Performed on Synechocystis 6803 / Thermocynechococcus elongatus

26. Seeing is believing Some ’’’assignments’’’ Glutamine synthase Phycobilisomefragment ATP synthase T-shapedparticle GroEl-GroES from the PDB site “molecule of the month displays”

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

28. Analysis of Photosystem II arrays and double dimers Phycobilisome model 16.7 nm 12.5 nm

29. Analysis of Photosystem II double dimers Double dimer model Is there a specific subunit involved in double dimer formation?

30. Models for the photosynthetic membrane