Surface Analysis of the Photosystem I Complex by Electron and Atomic Force Microscopy Dimitrios Fotiadis 1 , Daniel J. Mu È ller 1,3 , Georgios Tsiotis 4 Lorenz Hasler 1 , Peter Tittmann 5 , Thierry Mini 2 , Paul Jeno È 2 Heinz Gross 5 and Andreas Engel 1 * 1 M. E. Mu È ller Institute for Microscopy and 2 Division of Biochemistry Biozentrum of the University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland 3 Forschungszentrum Ju È lich Structural Biology D-52425 Ju È lich, Germany 4 Department of Chemistry University of Crete P.O. Box 1470, 71409 Iraklion Greece 5 Institute of Applied Physics Swiss Federal Institute of Technology, Ho Ènggerberg CH-8093 Zu È rich, Switzerland Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise de®nition of the lumenal and stromal PSI surfaces. The lumenal surface was com- posed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrin- sic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e 0 or were at the sites of putative a-helix-connecting loops estimated from the 4 A Ê 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; molecular dissection; 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-dependent transfer of electrons from reduced plastocyanin or cytochrome c 6 to soluble ferredoxin or ¯avodoxin across the thylakoid membrane. The functional reaction center of the thermophilic cyanobacterium Synechococcus sp. consists of 11 protein subunits and 90 chlorophyll molecules, assembled into a 340 kDa complex (Krauss et al., 1993; Golbeck, 1994). The two high molecular mass subunits PsaA and PsaB, 83 kDa each, are very hydrophobic and bind the electron transfer components P700, A 0 , A 1 ,F X , an unspeci®ed number of b-carotene mol- ecules and most of the chlorophylls. The other nine subunits (PsaC, -D, -E, -F, -I, -J, -K, -L and -M) are small, each having a mass below 20 kDa. The extrinsic protein PsaC (9 kDa) at the stromal side contains two [4Fe-4S]-clusters, the F A and F B centers, which are the terminal electron acceptors in the electron transfer chain (Oh-oka et al., 1987). Thus, the electron released by photo-oxidation of P700 at the lumenal side tunnels through the cas- cade A 0 ,A 1 ,F X to reach F A ,F B in about 500 ns (Brettel, 1997). PsaC appears to bind loosely to the PsaA/PsaB heterodimer in the absence of the other two extrinsic proteins, PsaD (16 kDa) and PsaE (8 kDa). A stable binding of PsaC to the PsaA/ E-mail address of the corresponding author: aengel@ubaclu.unibas.ch Abbreviations used: Mes, 2-(N-morpholino)- ethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)- piperazine-1-ethane-sulfonic acid; Pha, allophycocyanin subunit; AFM, atomic force microscopy; BA, benzamidine; Chl, chlorophyll; DMPC, dimyristoyl phosphatidylcholine; CAS, e-amino-n-caproic acid; A 0 , A 1 , electron acceptors; EM, electron microscopy; FRC, Fourier ring correlation function; F X ,F A ,F B , iron sulfur centers; 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