surface analysis of the photosystem i complex by electron and atomic force microscopy

Surface Analysis of the Photosystem I Complex byElectron and Atomic Force Microscopy

Dimitrios Fotiadis1, Daniel J. MuÈ ller1,3, Georgios Tsiotis4

Lorenz Hasler1, Peter Tittmann5, Thierry Mini2, Paul JenoÈ 2

Heinz Gross5 and Andreas Engel1*

1M. E. MuÈ ller Institute forMicroscopy and 2Division ofBiochemistry Biozentrum of theUniversity of BaselKlingelbergstrasse 70CH-4056 Basel, Switzerland3Forschungszentrum JuÈ lichStructural BiologyD-52425 JuÈ lich, Germany4Department of ChemistryUniversity of CreteP.O. Box 1470, 71409 IraklionGreece5Institute of Applied PhysicsSwiss Federal Institute ofTechnology, HoÈnggerbergCH-8093 ZuÈ rich, Switzerland

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction centerfrom Synechococcus sp. OD24 were analyzed by electron and atomic forcemicroscopy. Surface relief reconstructions from electron micrographs offreeze-dried unidirectionally shadowed samples and topographs recordedwith the atomic force microscope (AFM) provided a precise de®nition ofthe lumenal and stromal PSI surfaces. The lumenal surface was com-posed of four protrusions that surrounded an indentation. One of theprotrusions, the PsaF subunit, was often missing. Removal of the extrin-sic proteins with the AFM stylus exposed the stromal side of the PSIcore, whose surface structure could then be imaged at a resolution betterthan 1.4 nm. This interfacial surface between core and extrinsic subunits,had a pseudo-2-fold symmetry and protrusions that correlated with thesurface helices e and e0 or were at the sites of putative a-helix-connectingloops estimated from the 4 AÊ map of the complex. The molecular dissec-tion achieved with the AFM, opens new possibilities to unveil the inter-faces between subunits of supramolecular assemblies.

# 1998 Academic Press

Keywords: atomic force microscopy; electron microscopy; moleculardissection; photosystem I; two-dimensional crystals*Corresponding author

Introduction

Photosystem I (PSI) is one of the two pigment-containing reaction centers of oxygenic photosyn-thesis found in cyanobacteria and plants (Barber &Andersson, 1994). It catalyzes the light-dependenttransfer of electrons from reduced plastocyanin orcytochrome c6 to soluble ferredoxin or ¯avodoxin

across the thylakoid membrane. The functionalreaction center of the thermophilic cyanobacteriumSynechococcus sp. consists of 11 protein subunitsand 90 chlorophyll molecules, assembled into a340 kDa complex (Krauss et al., 1993; Golbeck,1994). The two high molecular mass subunits PsaAand PsaB, 83 kDa each, are very hydrophobic andbind the electron transfer components P700, A0,A1, FX, an unspeci®ed number of b-carotene mol-ecules and most of the chlorophylls. The other ninesubunits (PsaC, -D, -E, -F, -I, -J, -K, -L and -M) aresmall, each having a mass below 20 kDa.

The extrinsic protein PsaC (9 kDa) at the stromalside contains two [4Fe-4S]-clusters, the FA and FB

centers, which are the terminal electron acceptorsin the electron transfer chain (Oh-oka et al., 1987).Thus, the electron released by photo-oxidation ofP700 at the lumenal side tunnels through the cas-cade A0, A1, FX to reach FA, FB in about 500 ns(Brettel, 1997). PsaC appears to bind loosely to thePsaA/PsaB heterodimer in the absence of the othertwo extrinsic proteins, PsaD (16 kDa) and PsaE(8 kDa). A stable binding of PsaC to the PsaA/

E-mail address of the corresponding author:[email protected]

Abbreviations used: Mes, 2-(N-morpholino)-ethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-piperazine-1-ethane-sulfonic acid; Pha, allophycocyaninsubunit; AFM, atomic force microscopy; BA,benzamidine; Chl, chlorophyll; DMPC, dimyristoylphosphatidylcholine; CAS, e-amino-n-caproic acid; A0,A1, electron acceptors; EM, electron microscopy; FRC,Fourier ring correlation function; FX, FA, FB, iron sulfurcenters; LPR, lipid-to-protein ratio; SB-12, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; OTG,octyl-b-thioglucopyranoside; Psa, photosystem I protein;P700, primary electron donor; SSNR, spectral signal-to-noise ratio; 2D, two-dimensional; PS, photosystem.

Article No. mb982097 J. Mol. Biol. (1998) 283, 83±94

0022±2836/98/410083±12 $30.00/0 # 1998 Academic Press

PsaB core requires the supplementary binding ofPsaD to the complex (Zhao et al., 1990; Li et al.,1991). PsaD is also involved in the interactionbetween PSI and soluble ferredoxin during itsphotoreduction (Lelong et al., 1994), while PsaE isimportant for the cyclic electron transport (Zhaoet al., 1993), as well as for the interaction betweenthe terminal electron acceptor and ferredoxin(Rousseau et al., 1993; Sonoike et al., 1993).

The function of the PsaF protein (15 kDa) at thelumenal side has been subject to discussion. Inintact cells of the green alga Chlamydomonas rein-hardtii, PsaF is implicated in the electron transferfrom plastocyanin to oxidized P700 by providing adocking site for the electron donor: psaFÿ mutantsof this organism had a dramatically reduced elec-tron transfer rate (Farah et al., 1995). In contrast, apsaFÿ mutant of the cyanobacterium SynechocystisPCC 6803 exhibited normal electron transfer toP700�, implying that PsaF is not essential for thedocking of either cytochrome c6 or plastocyanin toPSI (Xu et al., 1994; Hippler et al., 1996). While PSIis extracted as a mixture of trimers and monomersfrom thylakoid membranes of wild-type cyanobac-teria, PSI from mutants that lack the PsaL protein(16 kDa) exists exclusively as a monomer aftermembrane solubilization (Chitnis & Chitnis, 1993).In addition, proteolysis studies have shown PsaLto be located about the 3-fold axis of the trimer,thus holding it together (Chitnis & Chitnis, 1993).Little is known about the function of the four othermembrane intrinsic subunits (PsaI, -J, -K and -M)that have molecular masses ranging from 3 to8 kDa (Golbeck, 1994).

A wealth of information about the PSI complexis available from different structure determinationmethods. Structural analysis of solubilized PSI byelectron microscopy (EM) has provided evidencefor the presence of trimeric complexes in the nativemembrane (Hladik & Sofrova, 1991; Tsiotis et al.,1995). Analysis of solubilized trimeric complexeshas allowed the positions of the subunits PsaC, -D,-E, -F and -J (Kruip et al., 1997), as well as the dock-ing sites for ferredoxin (Lelong et al., 1996) and ¯a-vodoxin (MuÈ hlenhoff et al., 1996) to be mapped.A three-dimensional reconstruction of negativelystained 2D crystals and a projection map of frozen-hydrated 2D crystals at 8 AÊ resolution (Karraschet al., 1996), have given precise information on theintegration of the PSI complex in the bilayer. Thethree-dimensional structure of PSI from the ther-mophilic cyanobacterium Synechococcus elongatusdetermined by X-ray crystallography is now avail-able at a resolution of 4 AÊ (Schubert et al., 1997).This model reveals 34 transmembrane and ninesurface a-helices, as well as the three [4Fe-4S] cen-ters and 89 chlorophyll molecules per monomer.

Nevertheless, detailed structural information onthe surface topography of this fascinating enzymeis lacking. This has prompted a study of PSI fromSynechocystis sp. PCC 6803 using domain-speci®cantibodies and proteolysis (Sun et al., 1997). Moredirect approaches to investigate the surface struc-

ture of proteins are provided by microscopy.Surface relief reconstructions from electron micro-graphs of unidirectionally shadowed 2D crystalshave yielded information to a resolution betterthan 1 nm (Walz et al., 1996). Topographs of mem-brane proteins in aqueous environment have beenacquired at a resolution better than 1 nm using theatomic force microscope (AFM; Karrasch et al.,1994; Schabert et al., 1995; MuÈ ller et al., 1995a)allowing single protein loops to be localized. Theability of the AFM to directly visualize protein con-formational changes at high-resolution has alsobeen demonstrated (MuÈ ller et al., 1995b, 1996).

Here, we present the lumenal and stromal topo-graphy of the PSI complex when reconstituted in alipid bilayer. The structural information of thenative PSI reaction center recorded with the AFMin buffer solution is compared with a surfacereconstruction from freeze-dried and unidirection-ally, heavy-metal shadowed PSI crystals recordedwith the electron microscope. In addition, wedemonstrate the feasibility of nanometer scale dis-sections using the AFM stylus, which gave us thepossibility of exploring otherwise hidden surfaces.

Results

The isolated and reconstituted PSI reaction cen-ter from the thermophilic cyanobacterium Synecho-coccus sp. clone OD24 yielded a protein complexconsisting of at least nine subunits, as illustratedby the high-resolution SDS/polyacrylamide gel inFigure 1. Lanes 1 and 2 show the polypeptide com-position of the puri®ed PSI before and after itsreconstitution in lipid bilayers. The 2D crystalswere pelleted prior to solubilization for SDS-PAGE, allowing the exact subunit composition ofthe PSI complexes that had been incorporated intomembranes to be determined (Figure 1, lane 2).The crystallized complexes exhibited essentiallythe same protein composition as the solubilizedPSI. The fuzzy band at 60 kDa is characteristic ofthe reaction center proteins PsaA and PsaB, whileseven distinct bands between 21.5 and 2.5 kDacould be assigned to the PsaD, -F, -L, -E, -C, -Kand -J subunits. All low molecular mass subunitsexcept PsaK could be identi®ed by mass spec-trometry (Table 1), while PsaK was assignedaccording to LuÈ neberg et al. (1994). In contrast tothe ®ndings reported by Karrasch et al. (1996), thePsaL subunit was still present in the complex afterthe reconstitution. The thin band above the 17 kDamarker was found to be a contamination by theallophycocyanin beta-subunit protein (PhaB,17 kDa; identi®ed by mass spectrometry, data notshown). Since 2D crystals were pelleted prior toSDS-PAGE, this band was only a weak shadow inlane 2. Depending on the preparation, anadditional sharp band below the 17 kDa markerwas found in the crystallized PSI. As a result of itsirreproducibility and weakness, this band couldnot be identi®ed by mass spectroscopy. Weak

84 Surface Structure of the Photosystem I Complex

bands, possibly corresponding to PsaM, and freepigments were visible below 2.5 kDa, while thethick band characteristic of lipids was observedonly in the reconstituted PSI. Laser-inducedabsorption changes at 817 nm demonstrated thatboth the solubilized and the crystallized PSI com-plexes were fully active (F. Drepper, personal com-munication).

Freeze-dried and unidirectionally metal-sha-dowed PSI 2D crystals (Figure 2) revealed rows ofmonomers that were integrated in the bilayer in analternating up and down manner as described byFord et al. (1990). The 2D crystals had a diameterof typically 2 mm (Figure 2a), and exhibited a dis-tinct surface corrugation (Figure 2b), resulting frommonomers packed in an orthorhombic lattice

with unit cell dimensions a � 13.8(�0.4) nm,b � 14.4(�0.3) nm (n � 28). Sharp diffraction spotsup to the reciprocal lattice order (6,0) indicated aresolution of about 2.4 nm (Figure 2c). Each unitcell contained two oppositely oriented monomers,a packing arrangement characteristic of p121 sym-metry, as illustrated by the average shown inFigure 2d. The resolution of such averages wasdetermined to either 1.4 nm by the Fourier ringcorrelation function (FRC; Saxton & Baumeister,1982) or to 1.5 nm by the spectral signal-to-noiseratio (SSNR; Unser et al., 1987). The distinct sha-dow (white arrowhead in Figure 2d) suggestedthat a fraction of one monomer protruded from themembrane, while the other monomer had a rather¯at surface. This protrusion has been assigned tothe stromal, water-soluble subunits PsaC, -D and-E (Ford et al., 1990).

Figure 3 shows an image of a mica-supportedPSI 2D crystal recorded in buffer solution (20 mMTris-HCl (pH 7.8), 300 mM KCl) with the AFMoperated in the de¯ection mode. The crystal latticeis distinct and seen to be embedded in a lipidbilayer. Besides such lipid bilayers with the PSIcomplex, two types of bilayers without PSI werefound. One was abundant, displaying a smoothand homogeneous structure with a height of4.1(�0.2) nm (n � 40). The other occurred less fre-quently, had a rough and inhomogeneous struc-ture with a thickness of 5.5(�0.3) nm (n � 40), andusually contoured the smooth lipid bilayers (datanot shown). In contrast, the thickness of PSI crys-tals adsorbed to mica was 11.0(�0.5) nm (n � 166)as result of the stromal extrinsic proteins forminghigh protrusions. These were clearly resolved intopographs of single layered crystals recorded athigh magni®cation (Figure 4a). While only fewprotrusions were missing in early scans (Figure 4a),their complete removal upon repetitive scanning ofthe same area was observed (Figure 4b). The highsignal-to-noise ratio of the topographs recorded inthe contact mode allowed this process to bedirectly monitored. It could be accelerated eitherby increasing the force applied to the stylus or by

Figure 1. Silver-stained SDS/polyacrylamide gel ofsolubilized (lane 1) and crystallized (lane 2) PSI reactioncenter from Synechococcus sp.

Table 1. Identi®cation of low molecular mass subunits of PSI from Synechococcus sp. by mass spectrometry

Subunit Mr (daltons) from sequence Mr (daltons) from es-ms Sequence of identified fragment

PsaD 1282.4 1282.1 SIGQNPNPSQLK1465.7 1465.3 IFPDGETVLIHPK1636.9 1636.4 EQVFEMPTAGAAVMR

PsaF 1472.6 1471.9 AAAAVNTTADPASGQK1018.1 1017.7 ELASGELTAK805.0 804.7 AYLIAVR

1223.5 1222.7 EIIIDVPLAIKPsaL 1151.3 1151.5 TFIGNLPAYRPsaE 745.9 745.8 YPVIVR

3151.4 3151.7 VNYTGYSGSASGVNTNNFALHEVQEVAPPK

PsaC 640.8 640.7 SMGLAY1009.1 1008.7 VYLGAETTR

PsaJ 1261.5 1261.2 FYPDLLFHPL

es-ms, electrospray mass spectrometry. The sequence fragments identi®ed in the database were from the organism Synechococcuselongatus, because Synechococcus sp. was reclassi®ed as Synechococcus elongatus (Fromme et al., 1994).

Surface Structure of the Photosystem I Complex 85

imaging at low salt concentration (e.g. 20 mM Tris-HCl (pH 7.8), 50 mM KCl). Since the other side ofthe PSI reaction centers (alternate rows in Figure 4aand b) remained essentially unchanged during

scanning, the presence of easily removeable pro-teins allowed the stromal side of the PSI complexto be unambiguously identi®ed. Three differentstates of the PSI complex with respect to its extrin-sic subunit composition could be detected reprodu-cibly (Figure 4, subframes 1, 2 and 3).

Most importantly, displacement of the wobbly,extrinsic subunits made the underlying surfacesavailable for structural analysis at high resolution.Figure 5a shows a crystalline area devoid of allextrinsic subunits. The stromal PSI sides, whichprotruded 3.5(�0.2) nm (n � 44; Figure 4a) out ofthe membrane before removal of the extrinsic pro-teins, now had a height of only 0.9(�0.2) nm(n � 99). In contrast, the lumenal side still pro-truded 1.7(�0.2) nm (n � 100) from the lipidbilayer. The power spectrum displayed in Figure 5bexhibits diffraction spots at 1.8 nm resolution.However, after correlation averaging the resolutioncalculated by the FRC was 1.2 nm, whereas theSSNR criterion yielded 1.4 nm. The standard devi-ation map simultaneously calculated (Schabert &Engel, 1994) exhibited values between 0.16 and0.29 nm in the region of the exposed stromal sur-face (data not shown). This surface was rathersmooth and characterized by a central narrowridge, which was subdivided into four distinctsmall domains (Figure 5c; white arrow), andextended from one corner of the complex to theother. This characteristic central ridge revealed apseudo-2-fold symmetry, marked in Figure 5c.Two smaller ridges protruded on both sides of thecentral ridge, running almost parallel with it.Another yet smaller protrusion was seen on onlyone side of the exposed stromal surface (Figure 5c,black arrowhead).

Figure 2. Two-dimensional PSI crystals shadowed unidirectionally with Ta/W at an elevation angle of 45�. a, Over-view of a 2D crystal after freeze-drying and unidirectional metal-shadowing at low magni®cation. b, High magni®-cation view of a shadowed crystal exhibiting periodic protrusions. c, The power spectrum of the same area, showingdiffraction spots up to the (6,0) order at 2.4 nmÿ1. d, Correlation average of b calculated from 508 motifs. The scalebars represent 500 nm (a), 50 nm (b), 5 nmÿ1 (c) and 5 nm (d). The arrows in a, b and d indicate the shadowingdirection, while the white arrowhead in d marks a pronounced shadow.

Figure 3. De¯ection mode image of a 2D PSI crystalrecorded with the AFM in buffer solution. The crystal-line sheet is surrounded by a lipid border. The averageheight of such crystals adsorbed to mica was11.0(�0.5) nm, while the lipid layer had a height of4.1(�0.2) nm. Imaging conditions: buffer solution,20 mM Tris-HCl (pH 7.8), 300 mM KCl, scan frequency5.1 Hz, applied force �500 pN. Heights were measuredfrom topographs recorded in the height mode. The scalebar represents 500 nm.


The lumenal surface of PSI was characterized byan approximately 0.9(�0.2) nm deep indentation,surrounded by an elliptical ring assembled fromelevations of different heights. Two high protru-sions (Figure 5c, broken black contour lines)extended by 1.7(�0.2) nm out of the membrane,and the two smaller ones (Figure 5c, continuousblack contour lines) by only 1.5(�0.2) nm. Becauseof the high signal-to-noise ratio such AFM imagesdocumented that the 2D crystals were composed oftwo PSI complex types with different intrinsic sub-unit compositions. The white arrowheads inFigure 5a mark reaction centers missing one of thefour lumenal protrusions. To calculate the corre-lation average displayed in Figure 5c, only unitcells exhibiting PSI complexes with complete lume-nal surfaces were used. The correlation averages 1(intact lumenal surface) and 2 (partial lumenal sur-face) in Figure 5 show unit cells calculated usingreferences of the two distinct forms of the lumenalside. Their difference map is displayed in Figure 5,subframe 3, and corresponds to a mass of 5 to10 kDa, assuming a protein density of 0.8 kDa/nm3. The fraction of these missing lumenal protru-sions depended on the batch of 2D crystals.Occasionally, however, such a protrusion wasfound to be sheared off by the AFM tip by multiplescanning of the same area at high magni®cation,while the other three lumenal protrusions did notchange. In contrast to the instantaneous displace-ment of the stromal subunits by scanning frames

of 4500 nm width (Figure 4a and b), lumenal pro-trusions were removed by the tip only rarely.

Figure 6 summarizes the PSI surface structuresacquired by electron (a) and atomic forcemicroscopy (b and c). The prominent, high lumenalelevation, sometimes missing on AFM topographs(Figure 5), is less pronounced on the electron-microscopical surface reconstruction (dotted con-tour line in Figure 6a). Features such as the inden-tation (white open arrowhead) on the lumenal sideand the adjacent protrusion (black arrowhead)could be detected in all three surface maps(Figure 6a, b and c). The stromal L-shaped protru-sion in the average calculated from AFM topo-graphs containing all extrinsic subunits (Figure 6b)had a height above the bilayer of 3.5(�0.2) nm. Itappeared to be more voluminous than the L-shaped protrusion of the surface relief recon-structed from three freeze-dried metal-shadowedcrystals (Figure 6a). The mass of the stromal pro-trusion removed by the tip of the AFM was esti-mated as 30 to 40 kDa from Figure 6b, which iscompatible with the removal of all extrinsic sub-units, PsaC, -D and -E. The former position of thisprotrusion is marked in Figure 6c by a continuouswhite line. A subdivision of the protrusion can bediscerned in Figure 6b, delineating one of the threewater-soluble proteins whose position is markedby the broken white contour line in Figure 6c. Thissubunit protruded by 2.6(�0.2) nm out of themembrane. The stromal surface freshly exposed onremoval of the extrinsic subunits is subdivided into

Figure 4. High-resolution AFM topograph of a 2D PSI crystal with different extrinsic subunit compositions. a, PSIcrystal showing different subunit compositions in the extrinsic, water-soluble proteins. A considerable fraction of theextrinsic proteins has already been removed during the zoom-in process. Whole complexes containing PsaC, -D and-E are marked by continuous circles, PsaE depleted PSI complexes are marked by broken circles, while reaction cen-ters depleted of PsaD are marked by squares (see the text). b, PSI crystal after almost complete removal of the extrin-sic, water-soluble proteins by repetitive scanning of the same area at high magni®cation; marks as in a. The threedifferent extrinsic complexes imaged are displayed at higher magni®cation in subframes 1 to 3. Imaging conditions:buffer solution, 20 mM Tris-HCl (pH 7.8), 300 mM KCl, scan frequency 5.1 Hz, applied force �100 pN. The scalebars in a and b represent 40 nm. The frame size of 1, 2 and 3 is 19 nm. The full gray level range is 4 nm in all topo-graphs.


®ve distinct, mostly elongated domains, the charac-teristic central ridge composed of four similar pro-trusions (Figure 6c, 1 and 2), two smaller ridges(Figure 6c, 3 and 4) and a small elevation(Figure 6c, 5). The smallest domain was close tothe 3-fold axis of the trimeric PSI form (asterisks inFigure 6).

Discussion

We have isolated and reconstituted functionalPSI complexes into 2D arrays. The solubilizedand crystallized PSI had essentially the same pro-tein composition, as shown by SDS/polyacryl-amide gels, whose bands could in most cases beidenti®ed by mass spectroscopy. The small differ-ences between the solubilized and the crystallinePSI observed in regions above and below the17 kDa marker persisted during several puri®-cations and reconstitution experiments. The bandabove the 17 kDa marker corresponding to PhaBin the solubilized PSI was largely eliminatedfrom 2D crystals by centrifugation. The additionalunidenti®ed thin band in the reconstituted PSIcomplexes located below the 17 kDa may be a

degradation product resulting from dialysis atelevated temperature. Since relatively low concen-trations of OTG were used in our isolation andreconstitution procedure, the loss of the PsaLsubunit occuring in previous 2D crystallizationexperiments was avoided (BoÈ ttcher et al., 1992;Karrasch et al., 1996).

Electron microscopy of unidirectionally sha-dowed crystals revealed features similar to thosereported by Ford et al. (1990) and BoÈ ttcher et al.(1992), but at higher resolution as the result of theimproved freeze-drying, metal-shadowing tech-nique employed (Gross et al., 1990). It is interestingto note the excellent qualitative agreement of the®nest structural features seen in the relief withthose of the 3D reconstruction from negativelystained samples (Karrasch et al., 1996). However,the notorious problem of handedness with such 3Dmaps should also be pointed out (Heymann et al.,1997). Here, we have used AFM topographs toestablish the handedness of the relief reconstruc-tions.

Two different types of lipid bilayers wereobserved in the AFM. We assign the abundant,smooth membranes to DMPC bilayers, becausetheir height, 4.1(�0.2) nm, is in good agreement

Figure 5. High-resolution AFM topograph of a 2D PSI crystal after removal of the extrinsic subunits. a, The whitearrowheads indicate PSI complexes, seen from the lumenal side, which have lost one of the four protrusions. Thefreshly exposed stromal surface of the PsaA/PsaB heterodimer exhibits a ®ne striation and protrudes 0.9(�0.2) nm(n � 99) out of the membrane. Imperfections in the crystal can be seen (black arrowheads). b, The calculated diffrac-tion pattern of a. The (2,8) order at 1.8 nmÿ1 is marked by a circle. c, Correlation average of unit cells with intactlumenal surfaces (two different images; averaged motifs � 302). A long central ridge is visible on the stromal side(white arrow). Two high lumenal protrusions (broken black contour lines) extended by 1.7(�0.2) nm (n � 100) out ofthe membrane, and two smaller ones (continuous black contour lines) by only 1.5(�0.2) nm (n � 100). 1, Unit cell ofthe intact lumenal surface (two different images; averaged motifs � 302). 2, Unit cell of the partial lumenal surface,where PsaF is missing (two different images; averaged motifs � 184). 3, Difference map of 1 and 2. Imaging con-ditions: buffer solution, 20 mM Tris-HCl (pH 7.8), 300 mM KCl, scan frequency 5.5 Hz, applied force �100 pN. Thescale bars represent 25 nm (a), 5 nmÿ1 (b) and 10 nm (c). The frame size in 1, 2 and 3 is that of one unit cell: 14.4 nm(horizontally) and 13.8 nm (vertically). The full gray level range of a is 2 nm and for c, 1, 2 and 3, 1.7 nm. Thepseudo-2-fold axis is marked in white.


with previous DMPC bilayer thickness measure-ments (Knoll et al., 1981; Johnson et al., 1991) aswell as with the DMPC thickness derived fromthe atomic model of porin OmpF reconstitutedinto 2D crystals (Schabert et al., 1995). The mini-mal lipid-to-protein ratio (LPR) required for 2Dcrystallization of PSI from Synechococcus sp. hasbeen reported as 0.25 (Ford et al., 1990; Karraschet al., 1996). However, the best crystallization con-ditions (Karrasch et al., 1996), which were alsoused here, required an LPR of 1. Consequently,an abundance of such smooth DMPC membraneswas to be expected. The less abundant lipidlayers with an inhomogeneous surface structureand a thickness of 5.5(�0.3) nm are likely to beendogenous lipids carried along by the solubil-ized PSI complexes.

When the same area of a 2D PSI crystal wasscanned repeatedly at high magni®cation, weobserved the complete removal of the weaklybound extrinsic, water-soluble proteins PsaC, -Dand -E. This gentle and precise molecular dissec-tion exposed the underlying surface of the PsaA/PsaB heterodimer. The intermediates in the subunitremoval steps, and the freshly exposed stromalsurface could all be imaged at high resolution.Accordingly, the structure of the interface betweenthe extrinsic and the intrinsic subunits of PSI onthe stromal side could be imaged under native con-ditions in buffer solution. Furthermore, the surfacetopographies obtained for the 2D PSI crystalsreconstituted in the presence of lipids, extend aprevious study on the integration of the complexin the lipid bilayer (Karrasch et al., 1996).

The stromal surface structure of the whole PSIreaction center shown in Figure 6a (EM) and b

(AFM), containing PsaC, -D and -E, agrees withthe macroscopic surface structure derived fromelectron microscopy (Karrasch et al., 1996; Kruipet al., 1997) and X-ray crystallography (Schubertet al., 1997). Two distinct molecular dissection stepscould reproducibly be detected during imagingwith the AFM, as illustrated in Figure 4, subframes2 to 3. The alignment of the L-shaped extrinsic pro-tein cluster of the macroscopic X-ray model of PSI(Schubert et al., 1997) with the correlation averageof the whole complex shown in Figure 6b, allowsthese steps to be interpreted. Accordingly, sub-frame 1 in Figure 4 shows the full extrinsic com-plex, while subframe 2 reveals removal of PsaE,and subframe 3 reveals displacement of PsaD. Thetotal mass removed was estimated as 30 to 40 kDafrom the average in Figure 6b, accounting for theloss of all extrinsic subunits. It is interesting to notethat the topographs recorded with the AFM reveala stromal protrusion that is considerably largerthan that of the surface relief reconstructionobtained by electron microscopy. This may beexpected for such a prominent corrugation con-toured with a tip of typically 2 nm radius(Schabert & Engel, 1994), and from the lateralmovements of this wobbly protrusion. The smalldisplacement of the stromal elevation induced bylateral forces during scanning was evaluated as1.0(�0.2) nm from the difference map of averagesfrom the trace and the retrace topograph. How-ever, because the optical lever is orders of magni-tude more sensitive in measuring vertical thanlateral forces, the latter could not be estimatedwhile operating the AFM at vertical forces around100 pN. In spite of the larger size of the stromalelevation, the mass estimated for this protrusion is

Figure 6. Summary of PSI surface structures. a, Surface reconstruction from unidirectionally shadowed PSI 2D crys-tals. b, Average of two independent AFM images containing all the extrinsic proteins (averaged motifs � 247).c, Average of three independent AFM images depleted of all extrinsic subunits (averaged motifs � 535). Asterisks ina, b and c mark the location of the 3-fold axis of the trimeric PSI form, while dotted contour lines indicate the lume-nal protrusion, which is sometimes missing (see the text and Figure 5). The stromal protrusion (extrinsic proteins) iscontoured by a continuous white line in c at its position before removal by the AFM stylus. The broken white linemarks the PsaD subunit, which is located on the stromal surface. The white open arrowheads indicate the indentationon the lumenal side and black arrowheads a pronounced elevation located just beside the indentation. The whitearrowheads mark the binding pocket for ferredoxin/¯avodoxin (Fromme et al., 1994; Lelong et al., 1996; MuÈ hlenhoffet al., 1996). Protrusions of the stromal core complex surface are outlined in white (1 to 5). The PSI surface structuresare displayed as reliefs tilted by 5�. The scale bars represent 10 nm. The full gray level range of a and b is 3.5 nm,and for c 1.7 nm.


quite close to the total mass of the PsaC, -D and -Esubunits (33 kDa). On the other hand, a stringentboundary condition for artifact-free relief recon-structions from electron micrographs is a completelack of shadows (Guckenberger, 1985). Since thestromal surface protrudes by 3.5(�0.2) nm out ofthe membrane, this prerequisite was not achieved(see the shadow marked by the white arrowheadin Figure 2d). Therefore, the dimensions of thestromal protrusion are probably underestimated inthe relief reconstruction. Finally, the white arrow-heads at the L-shaped stromal protrusion inFigure 6 mark the location of the binding pocketfor ferredoxin (Lelong et al., 1996) or ¯avodoxin(MuÈ hlenhoff et al., 1996) as determined by EM andmodeling (Fromme et al., 1994). This binding site isclose to the quasi-2-fold axis of the exposed stro-mal surface (Figures 5c and 6c).

Figure 7 shows the stromal surface withoutextrinsic subunits acquired with the AFM at a lat-eral resolution of 1.2 to 1.4 nm. According to thestandard deviation map, the vertical resolutionwas approximately 0.2 nm in this region. The rel-evant elements of the 4 AÊ X-ray model (Schubertet al., 1997) that play a role for the surface topogra-phy are superimposed. The two small ridges(Figure 6c, domains 3 and 4) running parallel withthe large central ridge, are located in the region ofthe stromal extension of helix d (d0) and the per-ipherial helix e (e0). The position and direction ofthese small ridges are the same as those of the per-ipherial helices e and e0 in the X-ray model. Theloops between helices b (b0) and c (c0) also contrib-ute to the domains 3 and 4. Because the stromalextensions of the a-helices j ( j0) and k (k0) pointtowards the pseudo-2-fold axis (Schubert et al.,

Figure 7. Comparison between the surface topography of PSI after removal of the extrinsic proteins and the 4 AÊ

X-ray model (Schubert et al., 1997). Distinct PSI subunits are marked by dotted lines. PsaA and PsaB subunits wereidenti®ed according to Sun et al. (1997) with a-helices labelled (a to k) belonging to PsaB and (a0 to k0) to PsaA. Thea-helices l, l0, m, m0, n, n0, o, o0, r, s, t, u, v and y were omitted. To enhance contrast, the AFM image was high-pass®ltered with a linear ramp and bandwidth limited to a resolution of 1.2 to 1.4 nm. The frame size is 14.2 nm (hori-zontally) and 12.1 nm (vertically). The full gray level range corresponds to 0.9 nm.


1997), the two protrusions of the central ridge thatare close to the pseudo-2-fold axis marked inFigure 5c, probably result from loops connectingthese helices. The two other protrusions of the cen-tral ridge that are located near helices g (g0) cannotbe unambiguously assigned to elements of the 4 AÊ

X-ray structure. Possible candidates responsible forthese elevations are the stromal extension of helixa0, the loops of helices f (f0) and g (g0) (Sun et al.,1997), and the loops of helices h (h0) and i (i0). Thesmallest domain (5 in Figure 6c) is related to thestromal extension of helix q of subunit PsaL. Thesmall elevation situated near helices w and x inFigure 7 is thought to be the stromal connection ofthese two a-helices, which belong to subunit PsaF(Schubert et al., 1997).

In independent experiments, the lumenal sur-face of the PSI complex exhibited a distinctindentation (Ford et al., 1990; BoÈ ttcher et al., 1992;Karrasch et al., 1996), which has been modeled asthe docking site for the electron donor, plastocya-nin (Fromme et al., 1994). This depression is nowseen at higher resolution in the presented surfacerelief reconstruction from electron micrographs(Figure 6a), as well as in topographs of nativePSI crystal surfaces (Figure 6b), and even moreclearly in topographs of PSI crystals after theremoval of their extrinsic subunits (Figure 6c).The indentation exhibits a depth of 0.9(�0.2) nm,which is in good agreement with the depth of 0.5to 1 nm estimated from the 3D reconstruction oftilted, negatively stained EM images (Karraschet al., 1996). This depression is surrounded byfour distinct protrusions, of which one is some-times missing (Figure 5). The location of themissing protrusion corresponds to that previouslyassigned to the PsaF/PsaJ subunits by electronmicroscopy of wild-type and PsaF/PsaJ-deletionmutants (Kruip et al., 1997). The mass estimatedfrom the difference map in Figure 5, subframe 3(5 to 10 kDa) corresponds to the hydrophilicdomain of PsaF calculated under the assumptionthat PsaF consists of two membrane-spanninga-helices and a hydrophilic lumenal domain. Theoccasional absence of the surface domain thusrelated to subunit PsaF in AFM topographs of2D PSI crystals and the observation of a less pro-nounced protrusion on the electron-microscopicalsurface reconstruction (dotted contour line inFigure 6a) suggests that PsaF or the heterodimerPsaF/PsaJ dissociates from the PSI complex priorto 2D crystallization. Furthermore, the elevationunder discussion can occasionally be pushedaway by the scanning tip, while the other threelumenal protrusions are not affected. This indi-cates that the peripherial subunit PsaF is lesswell anchored in the complex than the subunitsPsaA and PsaB that give rise to the more stableprotrusions.

The present work has provided precise infor-mation on the integration of the PSI reaction centerin the bilayer. Measurement of the heights for thestromal and lumenal protrusion from AFM topo-

graphs allowed the boundaries of the membrane-embedded complex to be de®ned. On the basis ofthese data, it should be possible to ®t the X-raymodel into a lipid bilayer to gain supplementaryinformation on interactions of the PSI complexwith the lipids.

Materials and Methods

Chemicals

Dimyristoyl phosphatidylcholine (DMPC) andN-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfo-nate (SB-12) were purchased from the Sigma ChemicalCo. (St Louis, MO). Octyl-b-D-thioglucopyranoside(OTG) was from Calbiochem Co. (La Jolla, CA). Allother chemicals used for membrane preparation, puri®-cation and reconstitution of PSI were of analytical grade.

Isolation of the PSI complexes

Cells of Synechococcus sp. clone OD24 were kindlyprovided by Drs M. Miller and R. P. Cox, Odense Uni-versity, Denmark. Thylakoid membranes for the isolationof PSI complexes were prepared as described (Tsiotiset al., 1993). The membranes, suspended in 20 mM Mes-NaOH (pH 6), 10 mM MgCl2, 2 mM KH2PO4, 1 mMbenzamidine (BA), 1 mM e-amino-n-caproic acid (CAS),500 mM mannitol, 0.01% NaN3 were incubated with0.1% SB-12 for 20 minutes at room temperature. This®rst solubilization step led to the removal of phycobili-somes. After centrifugation (40 minutes, 4�C, 117,000 gat rav, Beckman TLA-100 rotor) the pellet was resus-pended in the same buffer, to the starting volume.A second solubilization step, under the same conditionsas before, and a subsequent centrifugation, led to theextraction of essentially all Photosystem II. The resultingpellet was then resuspended in 20 mM Mes-NaOH(pH 6), 10 mM MgCl2, 2 mM KH2PO4, 1 mM BA, 1 mMCAS, 500 mM mannitol, 0.01% NaN3 to the startingvolume, solubilized in 1% SB-12 at 4�C for 50 minutes,and centrifuged. The supernatant was collected andloaded onto a sucrose density-gradient (10% to 30% (w/w) in Tris-buffer (10 mM Tris-HCl (pH 8), 1 mM BA,1 mM CAS, 0.01% NaN3), containing 0.4 % (w/v) OTGand spun for 16 hours at 4�C (160,000 g at rav, KontronTST 41.14 rotor). The gradient yielded an orange layer atthe top, and two green layers. The lower green band wascollected and stored at 4�C. To ensure a complete deter-gent exchange (SB-12 versus OTG) and to remove somesmall contaminants, the lower green band was loadedonto a Q-Sepharose Fast Flow (Pharmacia) column,washed with 0.4% (w/v) OTG in Tris-buffer (3 � thevolume of the column material) and eluted with thesame buffer enriched with 400 mM NaCl.

Reconstitution

Isolated PSI complexes were mixed with DMPC solu-bilized in Tris-buffer containing OTG to achieve an LPRof 1 (w/w). The ®nal protein concentration was adjustedto 1 mg/ml and the ®nal OTG content to 0.95% (w/v).The reconstitution mixture (120 ml) was dialyzed against10 mM Mes-NaOH (pH 6), 6 g/l ammonium ferricitrate(Karrasch et al., 1996), in a temperature-controlled, con-tinuous-¯ow dialysis apparatus (Jap et al., 1992). Thedialysis cell temperature was adjusted to 25�C for the®rst 24 hours and increased linearly to 37�C during the


next 12 hours. After 24 hours at 37�C, the temperaturewas decreased linearly to 25�C within 12 hours, giving atotal dialysis time of 72 hours.

SDS-PAGE

Solubilized or reconstituted PSI was incubated insample buffer containing 3.4% (w/v) SDS and 3.6% (v/v) b-mercaptoethanol for six hours at room temperature.The samples were run on 16% to 22.5% polyacrylamidegels (Laemmli, 1970) containing 6 M urea, and silver-stained.

Mass spectral analysis

Protein identi®cation was carried out by excising thebands of interest from a Coomassie brilliant blue R250-stained SDS/polyacrylamide gel and digestion of theseparated protein in the gel piece with trypsin (Hellmanet al., 1995). The resulting peptides were desorbed from a100 mm i.d. � 280 mm o.d. capillary column packed withPOROS R2 into the mass spectrometer with a ten minutegradient consisting of 0% to 80% (v/v) methanol, 0.05%(v/v) acetic acid. Peptides were sequenced on-line with adata-controlled scanning procedure (Stahl et al., 1996) ata collision gas pressure of 3 mTorr and a collision energyof ÿ32 eV. Spectra were recorded on a TSQ7000 triplequadrupole instrument (Finnigan, San JoseÂ, CA)equipped with a microscale electrospray interface (Daviset al., 1995). Tandem mass spectra were correlated withthe OWL non-redundant composite protein sequencedatabase using the SEQUEST database searching pro-gram (Yates et al., 1995).

Heavy-metal shadowing

Sample preparation and operation of the EM wereperformed as described (Walz et al., 1996). Images wererecorded with a Gatan 694 slow scan CCD camera at amagni®cation of 78,000� , corresponding to 0.31 nmpixel width, and directly used for correlation averaging(Saxton & Baumeister, 1982). The resolution limits of theimages were determined according to the Fourier ringcorrelation function (FRC; Saxton & Baumeister, 1982),and the spectral signal-to-noise ratio (SSNR; Unser et al.,1987). All digital image processing steps performed onthe unidirectionally metal-shadowed specimens werecarried out as described (Fuchs et al., 1995), using theMILAN software package. The three images employedfor the multiple-image reconstruction were selected sothat the difference between their azimuthal shadowingangles ranged between 90� and 150�. The handedness ofthe surface relief was adapted to that of the AFM topo-graphs.

Atomic force microscopy

Two-dimensional PSI crystal stock solution (1 mg/mlPSI in 10 mM Mes-NaOH (pH 6), 6 g/l ammonium ferri-citrate) was diluted 20-fold in 10 mM Hepes-NaOH(pH 7), 50 mM NaCl. A 20 ml drop of this solution wasdeposited on freshly cleaved muscovite mica (Mica NewYork Corp., New York) prepared as described (Schabert& Engel, 1994). After 30 minutes, the sample was gentlywashed with imaging buffer (20 mM Tris-HCl (pH 7.8),300 mM KCl) to remove membranes that were not ®rmlyattached to the support. High-resolution imaging wasperformed in contact mode at a force of �100 pN

applied to the tip. Forces were measured by calibratingforce curves with the nominal cantilever spring constant.A weak repulsion between tip and sample was achievedby adjusting the salt concentration (MuÈ ller & Engel,1997). Images were acquired with a commercial AFM(Nanoscope III, Digital Instruments, Santa Barbara, CA93117, USA) equipped with a 120 mm scanner (j-scanner)and a liquid cell. Oxide-sharpened Si3N4 cantilevers fromDigital Instruments with a length of 120 mm and a nom-inal spring constant of k � 0.38 N/m, or from Olympus(Tokyo, Japan) with a length of 100 mm and a force con-stant of k � 0.1 N/m, were used. Operation and cali-bration of the AFM was performed as described (MuÈ ller& Engel, 1997). Correlation averaging (Saxton &Baumeister, 1982) was carried out with the SEMPERimage processing system (Saxton et al., 1979). Images(512 � 512 pixels) were ¯attened line by line prior to cal-culating correlation averages using a reference unit cellselected from the raw data. The resolution of the calcu-lated averages was determined according to the Fourierring correlation function (FRC; Saxton & Baumeister,1982), and the spectral signal-to-noise ratio (SSNR; Unseret al., 1987). Simultaneously acquired trace and retraceimages were processed separately. The calculated corre-lation averages were subsequently added together tocompensate for friction effects (Schabert & Engel, 1994).The volume of the protrusions was calculated eitherfrom difference maps of averages or directly fromaverages by integrating the volume of 0.1 nm thick slicesabove a threshold that gives the appropriate contour ofthe protrusion. From this estimate, the mass of the pro-trusion was obtained using an average protein density of0.8 kDa/nm3.

Acknowledgments

This work was supported by the Swiss National Foun-dation for Scienti®c Research (grant 4036-44062 to A.E.).The authors gratefully acknowledge Dr F. Drepper at theAlbert-Ludwigs-UniversitaÈ t, Freiburg, Germany for per-forming the measurements of ¯ash-induced charge sep-aration and Drs M. Miller and R. P. Cox, OdenseUniversity, Odense, Denmark for providing cells of thecyanobacterium Synechococcus sp. clone OD24. We thankDr S. A. MuÈ ller for critical reading of the manuscript.

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Edited by W. Baumeister

(Received 22 May 1998; received in revised form 14 July 1998; accepted 15 July 1998)


surface analysis of the photosystem i complex by electron and atomic force microscopy

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