biophysics masters course 2002 1.photosynthetic membranes jan p. dekker

Biophysics Masters Course 2002

1. Photosynthetic Membranes

Jan P. Dekker

Contents

1a. Tubular membranes in Rb. Sphaeroides

1b. Grana membranes in green plants

Biophysics Masters Course 2002


Protein complexes in green plant thylakoid membranes

Organization of Photosystem II

PSII-LHCII supercomplex

1 = PSII-LHCII supercomplexes

2 = PSII core dimers3 = PSII core monomers4 = trimeric LHCII5 = monomeric LHC

1

2

3

4

5

Analysis of supercomplexes in grana membranes

Gel filtration chromatography Superdex 200 HR 10/60

PSII-LHCII supercomplex

1

2

3

4

5

6

1 = PSII membrane fragments2 = PSII-LHCII megacomplexes3 = PSII-LHCII supercomplexes4 = LHCII-CP29-CP24 complex5 = trimeric LHCII6 = monomeric LHC

Gel filtration chromatography Superdex 200 HR 10/60

Analysis of supercomplexes in grana membranes

Biophysical Technique:

Transmission Electron Microscopy

General Criterion for Resolution in MicroscopyGeneral Criterion for Resolution in Microscopy

Resolution =Resolution = 0.61 0.61 sin sin

0.61 0.61 (if sin (if sin 1) 1)

is the wavelength and is the wavelength and is the half opening angle of the magnifying is the half opening angle of the magnifying lenslens

Light MicroscopyLight Microscopy

Transmission Electron MicroscopyTransmission Electron Microscopy

Green light of 550 nm permits about 300 nm resolutionGreen light of 550 nm permits about 300 nm resolution

Wavelength Wavelength = = 1.221.22

EE1/21/2 0.004 nm for E = 100 keV0.004 nm for E = 100 keV

The practical resolution is about 0.1 nm because of lens The practical resolution is about 0.1 nm because of lens aberrationsaberrations

Primary electrons

X-rays

CathodeLuminescence

Specimen

Transmitted electrons

E

SecondaryElectrons (s.e.)

BackscatteredElectrons (b.s.e.)

Auger-electrons

AbsorbedElectrons

Electron-specimen InteractionsElectron-specimen Interactions

• 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

CathodeLuminescence

Specimen

Transmitted electrons

E

SecondaryElectrons (s.e.)

BackscatteredElectrons (b.s.e.)

Auger-electrons

AbsorbedElectrons

Two Types of Electron MicroscopesTwo Types of Electron Microscopes

• Elastic scattering: kinetic energy and Elastic scattering: kinetic energy and momentum (of the colliding electron and momentum (of the colliding electron and atom) are preservedatom) are preserved

• Inelastic scattering: kinetic energy is Inelastic scattering: kinetic energy is transferred to the specimen as internal transferred to the specimen as internal (not kinetic) energy(not kinetic) energy

Contrast arises from scattering of electrons Contrast arises from scattering of electrons by the specimenby the specimen

Two types of contrast arise from elastic Two types of contrast arise from elastic scatteringscattering• Scattering ContrastScattering Contrast• Phase Contrast Phase Contrast

Inelastically scattered electronsInelastically scattered electrons• Blur the image because of chromatic Blur the image because of chromatic

aberrationaberration• Cause radiation damage to the specimen Cause radiation damage to the specimen

Contrast in the TEMContrast in the TEM

Inelastic scattering (0-0.001 rad)Inelastic scattering (0-0.001 rad)radiation damageradiation damage

Elastic scattering (0-0.1 rad)Elastic scattering (0-0.1 rad)small angles: small angles: phase contrastphase contrastlarge angles: large angles: scattering contrastscattering contrast

Scattering of Electrons by an AtomScattering of Electrons by an Atom

Heavy elements scatter electron stronger Heavy elements scatter electron stronger than light elements: scattering increases than light elements: scattering increases with the atomic number Zwith the atomic number Z

The ratio elastic/inelastic scattering is The ratio elastic/inelastic scattering is proportional to Zproportional to Z

el./inel. = Z/19el./inel. = Z/19

So for So for light elementslight elements (carbon, nitrogen, (carbon, nitrogen, oxigen), oxigen), inelastic scatteringinelastic scattering is predominant, is predominant,for for heavy elementsheavy elements (uranium, tungsten, (uranium, tungsten, platinum, osmium) platinum, osmium) elastic scatteringelastic scattering is is predominantpredominant

• Inelastic scattering ~ ZInelastic scattering ~ Z1/31/3

• Elastic scattering ~ ZElastic scattering ~ Z4/34/3

Scattering of Electrons by an AtomScattering of Electrons by an Atom

• Scattered electronsScattered electrons– elasticelastic– inelasticinelastic

• 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 Ejection of atoms (knock-on

damage)damage)

Result:Result:

Conclusion:Conclusion:Do not pre-irradiate samples unnecessaryDo not pre-irradiate samples unnecessary

Interaction of fast Electrons with MatterInteraction of fast Electrons with Matter

Electron microscopy

Electron micrograph

PSI-300 topviewPSI-300 sideview

Contamination

Biophysical Technique:

Image Analysis

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.

Single Particle Image AnalysisSingle Particle Image Analysis

• pretreatment of projections pretreatment of projections - normalization of - normalization of densities within a maskdensities within a mask

• alignment of projectionsalignment of projections- rotational + translational shifts- rotational + translational shifts

• sorting of projectionssorting of projections-multivariate statistics + -multivariate statistics +

classificationclassification• calculation 2D projectioncalculation 2D projection

- summing of projections into - summing of projections into classesclasses

• calculation 3D structurecalculation 3D structure-combination of 2D projections-combination of 2D projections

• pretreatment of projections pretreatment of projections - normalization of - normalization of densities within a maskdensities within a mask

• alignment of projectionsalignment of projections- rotational + translational shifts- rotational + translational shifts

• sorting of projectionssorting of projections-multivariate statistics + -multivariate statistics +

classificationclassification• calculation 2D projectioncalculation 2D projection

- summing of projections into - summing of projections into classesclasses

• calculation 3D structurecalculation 3D structure-combination of 2D projections-combination of 2D projections

five main stepsfive main steps

Selected single particle projectionsSelected single particle projections

A gallery of rectangular supercomplexes of Photosystem II. One digital image file may contain a row of thousands of such images

Pretreatment of projections Pretreatment of projections (masking)(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.

Alignment procedure for randomly Alignment procedure for randomly oriented objectsoriented objects

rotationalrotationalalignmentalignment

translationaltranslationalalignmentalignment

Rotational correlationRotational correlationfunctionfunction

Cross correlationCross correlationfunctionfunction

imageimage referencereference

aligned imagealigned image

referencereference rotationallyrotationallyaligned imagealigned image

FFTFFT FFTFFT

FFTFFT FFTFFT

Alignment of projections Alignment of projections (rotational (rotational +translational)+translational)

Averaging of aligned projections Averaging of aligned projections

44 88 64641616 3232

128128 256256 512512 10241024 20482048

Description of image variationDescription of image variationfinding trends in density patternsfinding trends in density patterns

example: a 2-pixel imageexample: a 2-pixel image

technique:technique:

Multivariate Statistical AnalysisMultivariate Statistical Analysis

Eigenvector-Eigenimage Eigenvector-Eigenimage decompositiondecomposition

determination of image variation by compression of determination of image variation by compression of raw (“noisy”) dataraw (“noisy”) data

results:results:

description of individual mages by a linear description of individual mages by a linear combination ofcombination of

a limited number (“couple of dozen”) of a limited number (“couple of dozen”) of eigenimageseigenimages

images can be presented in a multidimensional images can be presented in a multidimensional vector spacevector space

close relatedness in space = close similarityclose relatedness in space = close similarity

Classification of PSII supercomplexes

SSMM SS

SS SS

SS

SS

SS

MM SS

SS MM SS

SS

LL

SS

SS

SS

SSLL

MM

MMLL

MM SS

SS

SS

SS

SS

SS

LL

LLMM

MM

Core complexCore 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].

LL

LLSS

SS

MM

MM

Zouni et al., Nature 409, 2001, 739-743

EM analysis of megacomplexes

Arabidopsis

Spinach

Megacomplexes are dimeric supercomplexes. They show how two supercomplexes can be attached to eachother.

Discovery of a multimer of LHCII present at low-frequency in solubilised thylakoid membranes

single particlesaveragedimages

Interpretation:a multimer containing 7copies of a LHCII trimer

Dekker et al., FEBS Lett. 449, 1999, 211

Protein complexes in green plant thylakoid membranes

EM analysis of grana membranes


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.

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].

Final results of imageanalysis of the large-spaced andsmall-spaced crystals. (a) and (c)The sums of 900 and 100 fragmentsof both types of crystals. The unitcells of both crystal types are indi-cated. Images are presented in theirmirror-versions, to facilitate com-parison with all previously pub-lished supercomplex structures. In(b) and (d), supercomplexes of theC2S2 type have been fitted into thelattices, to indicate the position ofthe innermost part of the peripheralantenna (one S LHCII trimer plusone CP26 and one CP29 subunit; ingreen) around the dimeric core part(in blue). The results suggest thatmost lattices have a C2S2M repeatingunit and that the minor lattice in Dhas a C2S2 repeating unit. From [2].

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

Arabidopsis

Arabidopsis membranes have a C2S2M2 repeating unit, alsoin a mutant with an antisense inhibition of CP26.


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].


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

biophysics masters course 2002 1.photosynthetic membranes jan p. dekker

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