1. Biophysics Masters Course 2002 1.Photosynthetic Membranes Jan P. Dekker

2. Contents 1a. Tubular membranes in Rb. Sphaeroides 1b. Grana membranes in green plants

3. Biophysics Masters Course 2002 1b. Grana membranes in green plants

4. Protein complexes in green plant thylakoid membranes

6. Organization of Photosystem II

7. PSII-LHCII supercomplex 1 = PSII-LHCII supercomplexes 2 = PSII core dimers 3 = PSII core monomers 4 = trimeric LHCII 5 = monomeric LHC 1 2 3 4 5 Analysis of supercomplexes in grana membranes Gel filtration chromatography Superdex 200 HR 10/60

8. PSII-LHCII supercomplex 1 2 3 4 5 6 1 = PSII membrane fragments 2 = PSII-LHCII megacomplexes 3 = PSII-LHCII supercomplexes 4 = LHCII-CP29-CP24 complex 5 = trimeric LHCII 6 = monomeric LHC Gel filtration chromatography Superdex 200 HR 10/60 Analysis of supercomplexes in grana membranes

9. Biophysical Technique: Transmission Electron Microscopy

10. General Criterion for Resolution in Microscopy Resolution = 0.61 0.61 sin   0.61 (if sin   1) is the wavelength and  is the half opening angle of the magnifying lens is the wavelength and  is the half opening angle of the magnifying lens Light Microscopy Transmission Electron Microscopy Green light of 550 nm permits about 300 nm resolution Wavelength = 1.22 E 1/2  0.004 nm for E = 100 keV The practical resolution is about 0.1 nm because of lens aberrations

11. Primary electrons X-rays Cathode Luminescence Specimen Transmitted electrons E Secondary Electrons (s.e.) Backscattered Electrons (b.s.e.) Auger-electrons Absorbed Electrons Electron-specimen Interactions

12. Scanning Electron Microscope (SEM) –Secondary Electrons –Back-scattered Electrons –(X-rays) Transmission Electron Microscope (TEM) –Transmitted Electrons –(X-rays) Scanning Electron Microscope (SEM) –Secondary Electrons –Back-scattered Electrons –(X-rays) Transmission Electron Microscope (TEM) –Transmitted Electrons –(X-rays) Primary electrons X-rays Cathode Luminescence Specimen Transmitted electrons E Secondary Electrons (s.e.) Backscattered Electrons (b.s.e.) Auger-electrons Absorbed Electrons Two Types of Electron Microscopes

13. Elastic scattering: kinetic energy and momentum (of the colliding electron and atom) are preservedElastic scattering: kinetic energy and momentum (of the colliding electron and atom) are preserved Inelastic scattering: kinetic energy is transferred to the specimen as internal (not kinetic) energyInelastic scattering: kinetic energy is transferred to the specimen as internal (not kinetic) energy Contrast arises from scattering of electrons by the specimen Two types of contrast arise from elastic scattering Scattering ContrastScattering Contrast Phase ContrastPhase Contrast Inelastically scattered electrons Blur the image because of chromatic aberrationBlur the image because of chromatic aberration Cause radiation damage to the specimenCause radiation damage to the specimen Contrast in the TEM

14. Inelastic scattering (0-0.001 rad) radiation damage Elastic scattering (0-0.1 rad) small angles: phase contrast large angles: scattering contrast Scattering of Electrons by an Atom

15. Heavy elements scatter electron stronger than light elements: scattering increases with the atomic number Z The ratio elastic/inelastic scattering is proportional to Z el./inel. = Z/19 So for light elements (carbon, nitrogen, oxigen), inelastic scattering is predominant, for heavy elements (uranium, tungsten, platinum, osmium) elastic scattering is predominant Inelastic scattering ~ Z 1/3Inelastic scattering ~ Z 1/3 Elastic scattering ~ Z 4/3Elastic scattering ~ Z 4/3 Scattering of Electrons by an Atom

16. Scattered electronsScattered electrons –elastic –inelastic Secondary electronsSecondary electrons Emission of X-raysEmission of X-rays Emission of visible lightEmission of visible light Temperature riseTemperature rise IonisationIonisation Bond breakageBond breakage Ejection of atoms (knock-on damage)Ejection of atoms (knock-on damage) Result: Conclusion: Do not pre-irradiate samples unnecessary Interaction of fast Electrons with Matter

17. Electron microscopy

18. Electron micrograph PSI-300 topview PSI-300 sideview Contamination

19. Biophysical Technique: Image Analysis

20. On the image as in the lower right corner randomly generated noise has been added; resulting in projections like the one in the top left corner. If such projections are summed in increasing number, the noise gradually fades out. The noise as observed in the electron microscopy pictures is very similar in strength as shown in this simulation.

21. Single Particle Image Analysis pretreatment of projections - normalization of densities within a maskpretreatment of projections - normalization of densities within a mask alignment of projections - rotational + translational shiftsalignment of projections - rotational + translational shifts sorting of projections -multivariate statistics + classificationsorting of projections -multivariate statistics + classification calculation 2D projection - summing of projections into classescalculation 2D projection - summing of projections into classes calculation 3D structure -combination of 2D projectionscalculation 3D structure -combination of 2D projections pretreatment of projections - normalization of densities within a maskpretreatment of projections - normalization of densities within a mask alignment of projections - rotational + translational shiftsalignment of projections - rotational + translational shifts sorting of projections -multivariate statistics + classificationsorting of projections -multivariate statistics + classification calculation 2D projection - summing of projections into classescalculation 2D projection - summing of projections into classes calculation 3D structure -combination of 2D projectionscalculation 3D structure -combination of 2D projections five main steps

22. Selected single particle projections A gallery of rectangular supercomplexes of Photosystem II. One digital image file may contain a row of thousands of such images

23. Pretreatment of projections (masking) A circular mask has been placed around each particle, within the mask the average density has been made zero and the contrast variance has been normalized to facilitate better comparison.

24. Alignment procedure for randomly oriented objects rotationalalignment translationalalignment Rotational correlation function Cross correlation function image reference aligned image reference rotationally rotationally aligned image FFT FFT FFT FFT

25. Alignment of projections (rotational +translational)

26. Averaging of aligned projections 4 86416 32 128256 512 10242048

27. Description of image variation finding trends in density patterns example: a 2-pixel image

28. technique: Multivariate Statistical Analysis Eigenvector-Eigenimage decomposition Eigenvector-Eigenimage decomposition determination of image variation by compression of raw (“noisy”) data results: description of individual mages by a linear combination of a limited number (“couple of dozen”) of eigenimages images can be presented in a multidimensional vector space close relatedness in space = close similarity

29. Classification of PSII supercomplexesSM SSSS S S M SS MSS L SS SSL M M L M S S SSS S L L M M Core complex A further type of variation found in many datasets: a slight tilt of the projection due to roughness of the carbon support film and/or of the surface of the particle. Almost all PSII complexes are, however, lying on their flat stromal surface and have their lumenal protrusions of extrinsic proteins facing upwards. From [1].

30. LL SS MM Zouni et al., Nature 409, 2001, 739-743

31. EM analysis of megacomplexes Arabidopsis Spinach Megacomplexes are dimeric supercomplexes. They show how two supercomplexes can be attached to eachother.

32. Discovery of a multimer of LHCII present at low- frequency in solubilised thylakoid membranes single particles averaged images Interpretation: a multimer containing 7 copies of a LHCII trimer Dekker et al., FEBS Lett. 449, 1999, 211

33. Protein complexes in green plant thylakoid membranes

34. EM analysis of grana membranes

35. Electron micrographs of two paired grana membrane fragments from spinach, negatively stained with 2% uranyl acetate. From the positions of the stain-excluding subunits, which presumably originate from the ex-trinsic proteins involved in oxygen evolution and which are attached to the core parts of PS II, it can be deduced that the membranes in (A) have a relative low ordering of the PS II core and that those in (B) show a semi-crystalline lattice in which the distance between rows of PS II complexes is about 26.3 nm. The two membranes overlap almost totally, but some small areas which are single layered can be recognized from a different staining pattern.

36. Electron micrographs of two paired grana membrane fragments from spinach, negatively stained with 2% uranyl acetate (A,B) or frozen-hydrated without stain (C). Asterisks indicate smooth areas where PSII is absent. The arrows indicate rows of PSII core particles in the upper and lower membranes. From [2].

37. Final results of image analysis of the large-spaced and small-spaced crystals. (a) and (c) The sums of 900 and 100 fragments of both types of crystals. The unit cells of both crystal types are indi- cated. Images are presented in their mirror-versions, to facilitate com- parison with all previously pub- lished supercomplex structures. In (b) and (d), supercomplexes of the C2S2 type have been fitted into the lattices, to indicate the position of the innermost part of the peripheral antenna (one S LHCII trimer plus one CP26 and one CP29 subunit; in green) around the dimeric core part (in blue). The results suggest that most lattices have a C2S2M repeating unit and that the minor lattice in D has a C2S2 repeating unit. From [2].

38. Model of the main repeating unit in spinach. From [2].

39. Arabidopsis

40. Arabidopsis membranes have a C2S2M2 repeating unit, also in a mutant with an antisense inhibition of CP26. EM analysis of grana membranes

41. Analysis of positions of PSII supercomplexes in the two layers of paired membranes with large-spaced crystalline macrodomains that show a relatively high level of ordering of the PSII supercomplexes. The black dots indicate the positions of central supercomplexes in both layers as found by alignment procedures. On these positions, rows of PSII complexes belonging to the lower membrane (in blue) or upper membrane (in red) have been fitted. The inner part of the peripheral antenna of the supercomplexes is indicated in green and yellow, respectively. From [2].

42. 1b. Grana membranes in green plants Literature: 1.E.J. Boekema, H. van Roon, F. Calkoen, R. Bassi, J.P. Dekker (1999) Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Biochemistry 38, 2233-2239 2.E.J. Boekema, J.F.L. van Breemen, H. van Roon, J.P. Dekker (2000) Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membranes of green plants. J. Mol. Biol. 301, 1123-1133