Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and
their interaction with the membrane☆
Ana A. Artenia,b, Ghada Ajlanib, Egbert J. Boekemaa,⁎
aDepartment of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
bInstitut de Biologie et de Technologies de Saclay, Centre National de la Recherche Scientifique, Commissariat à l'Energie Atomique, 91191 Gif-sur-Yvette, France
a b s t r a c t a r t i c l ei n f o
Received 14 November 2008
Received in revised form 14 January 2009
Accepted 15 January 2009
Available online 22 January 2009
Single particle image analysis
a giant, multi-subunit pigment–protein complex. This complex is composed of heterodimeric phycobili-
proteins that are assembled with the aid of linker polypeptides such that light absorption and energy
transfer to photosystem II are optimised. In this work we have studied, using single particle electron
microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin
hexamers. The images presented give much greater detail than those previously published, and in the best
two-dimensional projection maps a resolution of 13 Å was achieved. As well as giving a better overall picture
of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin
trimers within the core. Insights are gained into the attachment of this core to the membrane surface,
essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes
with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within
the complex at the rod-core interface.
© 2009 Elsevier B.V. All rights reserved.
Cyanobacteria and chloroplasts are defined by their ability to
carry out oxygenic photosynthesis, using two highly-conserved
photosystems located in the thylakoid membrane. In place of the
intra-membranous antennae found in most chloroplasts (and some
cyanobacteria), light-harvesting in cyanobacteria and several groups
of eukaryotic algae is principally performed by giant soluble
complexes associated with the membrane surface, called phycobili-
somes (PBS). Although a PBS is composed of hundreds of phyco-
biliproteins plus a few linker polypeptides, light energy absorbed
anywhere within the particle is efficiently transferred towards the
photosystems within the membrane .
A phycobiliprotein is composed of two polypeptide subunits
(α and β), each covalently binding an open-chain tetrapyrrole
chromophore known as a phycobilin. The αβ heterodimer assembles
into disc-like trimers (αβ)3 or hexamers (αβ)6, which possess a
central cavity. Linker polypeptides are believed to bind in the central
cavity of the phycobiliprotein discs [2,3]. The core of the PBS,
composed of trimeric (αβ)3discs of allophycocyanin (AP) stacked
into cylinders, forms a physical connection with the stromal surface
of the photosynthetic membrane. A series of rods, comprising cylin-
ders of stacked hexameric (αβ)6discs, radiate from the core. Different
linkers are specifically responsible for each level of phycobiliprotein
assembly and function to stabilise the PBS and optimise its absorp-
tion and energy transfer characteristics (Fig. 1).
High-resolution X-ray diffraction structures have been deter-
mined for various types of PBS components. For example, in the
crystal structure of the allophycocyanin core protein from the red
alga Porphyra yezoensis two trimers are loosely stacked together in
the unit cell to form a disc of diameter 105.3 Å and thickness 63.1 Å
. C-phycocyanin from the cyanobacterium Synechococcus elongatus
is a hexameric disc of 110 Å diameter by 60 Å thickness . While
many isolated components of different PBSs have been crystallised,
the structure of the entire PBS and the manner of its association with
photosystem II can only be suggested. Based on the overall structure
of PBSs as visualized by electron microscopy (EM), several distinct
morphological PBS families have been described. The most common
and best-described family is the hemi-discoidal PBSs, found in several
cyanobacteria, rhodophytes and the glaucophyte Cyanophora para-
doxa . Another family comprises the hemi-ellipsoidal PBSs, which
appear to have a substantially larger width. They were originally
found in the red alga Porphyridium cruentum , and are also present
in some cyanobacteria. A unique bundle-shaped PBS has been
described for the cyanobacterium Gloeobacter violaceus [8,9].
The hemi-discoidal PBS family has been further divided into three
subgroups according to structural differences of the core domain .
Biochimica et Biophysica Acta 1787 (2009) 272–279
Abbreviations: EM, electron microscopy; AP, allophycocyanin; PC, phycocyanin; PBS,
phycobilisome; LX, linker polypeptide located at position X of the PBS, where X can be C
(core), R (rod), RC (rod core) or CM (core membrane)
☆ Linkers with identical location are distinguished by a superscript just above the X
indicating their molecular mass.
⁎ Corresponding author. Tel.: +31 50 3634225; fax: +31 50 3634800.
E-mail address: email@example.com (E.J. Boekema).
0005-2728/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbabio
This domain is composed of 2, 3 or 5 cylinders of stacked allophy-
cocyanin trimers. Bi-cylindrical cores are exemplified by Synecho-
coccus sp. PCC 6301 . Tri-cylindrical cores have been observed
in several cyanobacteria such as Synechococcus sp. PCC7002 ,
Synechocystis spp. PCC6701 and PCC6803 [12,13], but are also
found in the red algae Porphyridium cruentum . Penta-
cylindrical cores, as exemplified by Mastigocladus laminosus and
Anabaena sp. PCC7120 [15,16], appear to be less common.
Each of the cylinders belonging to the core substructure is
composed of four trimeric AP discs (apart from the extra two cylin-
ders in penta-cylindrical cores which have only two discs), and these
discs have slightly different compositions (Fig. 1). The upper cylinder
(not present in bi-cylindrical cores) is composed of two simple AP
trimers (αβ)3that we will call T, and two other discs we designate T8
(T plus LC, a linker of 8 kDa). The two basal cylinders are composed of
one T disc, one T8 disc, one disc we designate B8 (T8 with one α-
subunit replaced by an isoform called AP-B) and one M disc (T with
one αβ heterodimer replaced by a red-shifted β isoform, β18, plus a
domain of the linker LCMfor α) . The core-membrane linker LCM,
the major linker polypeptide in the core, is responsible for the
assembly of AP discs into cylinders, and of cylinders together to form
the core. LCM also plays a key role in anchoring the PBS to the
photosynthetic membrane (possibly via direct interaction with PSII),
and in tuning the properties of the bound pigment cofactors
(phycobilins) such that absorbed light energy is funnelled towards
the photosystems. Two copies of this multifunctional polypeptide
(mass 75–125 kDa) are present per PBS core. The LCMpolypeptide is
divided into several domains (Fig. 1). The C-terminal portion of the
LCMcontains two to four repeated domains (Rep), depending on the
organism, each of about 120 residues. The Reps are ∼60% similar to
the conserved domains of the rod linkers (LR) and are likely to play
the same role (i.e. interaction between discs in a cylinder) . The
number of Reps determines the cylinder number in the core struc-
ture. The N-terminal portion of LCM(the PB domain) is 55% similar to
the AP α-subunit — it is this domain that replaces one α-subunit in
the M disc (see above). The PB domain is interrupted by the PB-loop,
which is about 50 to 65 residues long, depending on the organism
. The PB domain of the LCMis thus regarded as one of the core
subunits with its PB-loop protruding from the PBS core. Assembly
and function of the core can occur in the absence of the PB-loop,
leading to a model in which this loop is floating on one side of the
PBS, at the membrane surface . Finally, it has been proposed that
the domain of LCMcalled arm2 may be responsible for attachment of
the core to the membrane .
As mentioned above, the number of PBS core cylinders varies
according to the organism. EM studies indicate that each cylinder has
a diameter of about 110 Å while its length is given by the stacking of
two to four discs of thickness ∼30 Å. Additional cylinders called rods
radiate upwards from the core structure. The diameter of the discs
making up a rod is comparable to those of the core discs – 110 to
120 Å – but their thickness is typically about 60 Å, indicating that the
basic structural unit is a hexamer. In addition to the variation in core-
cylinder arrangement, the number and composition of rods attached
to the core can also vary. This number is usually between six and
eight, each rod being composed of 2 to 4 discs depending on the
species. Both of these parameters strongly influence the PBS mor-
phology. A hexameric phycocyanin (PC) disc is always located at the
rod-core linkage position, while the more distal complexes may be
PC, phycoerythrocyanin or phycoerythrin, depending on the organ-
ism and the growth conditions. EM and biochemical studies have led
to a number of models for the structure of the entire PBS, with
variation in the arrangements and associations of rods, and between
rods and the core . One of the most popular models of the hemi-
discoidal PBS has rods radiating out from the core with approxi-
mately even spacing [22,23]. PBSs from several cyanobacterial strains
can also contain 1 to 2 molecules of the enzyme Ferredoxin:NADP+
oxidoreductase (FNR) [24,25]. This enzyme catalyses electron transfer
between ferredoxin and NADP. In Synechocystis sp. strain PCC 6803,
two FNR isoforms are present: FNRS, similar in size to the plastid
FNR; and FNRL, containing an extra N-terminal domain, which is
similar to the 10 kDa rod-linker, CpcD (or LR
responsible for FNR binding to the PBS rods , and the FNRL
isoform is present in most PBS-containing cyanobacteria. There is,
however, some controversy over the location of FNRLbinding — to
the PC rod discs proximal  or distal  to the core.
In this paper we have used EM in combination with single
particle averaging to examine negatively-stained PBS particles from
WT Synechocystis as well as from a number of mutant strains.
Firstly the CK mutant was used, in which the PBSs lack PC rods and
do not bind FNR (Ughy and Ajlani, unpublished). Secondly the CB
mutant was exploited, where PBSs contain only one PC hexamer per
rod but retain FNRLin amounts similar to the wild type . We
also examined CBFS , having CB-like PBSs (one PC hexamer per
rod) but without FNRL. Finally recombinant FNRLwas reconstituted
back into this latter PBS in vitro. In the best two-dimensional
projection maps of truncated PBS, a resolution of 13 Å was achieved
and new densities were observed that could mediate interaction of
the cores with the membrane. By comparison of images of PBSs
10). This domain is
Fig.1. Representation of a hemi-discoidal PBS, as seen from side (A) and from the thylakoid membrane upwards (B). The architecture exhibits a tri-cylindrical core, from which
radiate six rods composed of three PC hexamers each. Differential colouring is used for the subunits of the 3 core cylinders in (B), to illustrate their structural organisation. The 2
core-membrane linkers LCMare represented, with their different domains: the PB-domain (forming part of the M discs), the PB-loop, arm2, and the Reps (filled ellipses).
Triangles represent the small core and rod linkers LCand LR
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279
with and without FNR, we suggest a possible localisation of FNR
within the PBS.
2. Materials and methods
2.1. Bacterial growth conditions
Wild-type and mutant strains of the cyanobacterium Synecho-
cystis sp. strain PCC 6803 were grown photoautotrophically in
modified Allen's medium in an illuminated orbital incubator at
32 °C, in a CO2-enriched atmosphere and under continuous light
(40 μE m−2s−1) [27,29].
2.2. PBS isolation and analysis
Cells were broken by vortexing with glass beads. PBSs were
prepared from Synechocystis sp. strain 6803 as described in [30,31].
Proteins were analysed by SDS-polyacrylamide gel electrophoresis on
12% Tris–Tricine gels. The OD at 620 nm was used to ensure appro-
ximately equal loading of different PBS samples; about 0.3 ODmL at
620 nm (100 μl at OD620=3≈100 μg of protein) was loaded per well.
PBS-containing samples were concentrated by precipitation with 10%
(v/v) trichloroacetic acid prior to loading. Proteins were visualized
using Coomassie blue stain. Recombinant FNRL(a gift from Dr. B.
Lagoutte) was used in reconstitution experiments, performed as in
, with PBS from the CBFS strain.
2.3. Transmission electron microscopy and image analysis
PBS preparations were negatively stained with 2% uranyl acetate
by the droplet method, after glutaraldehyde fixation. Drops of about
5 μL, containing the samples at 0.05 OD in 1 M potassium phosphate
pH 8 were deposited on the surface of glow-discharged and carbon-
coated copper grids. After 1 min the drop was removed and the grid
washed with the same buffer. Then a 5 μL drop of 0.5% glutaraldehyde
in 900 mM potassium phosphate was added to the residual material.
After 5 min fixation, the drop was washed 3 times in ammonium
acetate buffers (500, 50 and 10 mM, respectively), and stained with
2% uranyl acetate.
Electron microscopy was performed on a Philips CM120 electron
microscope (FEI, Eindhoven, The Netherlands) operated at 120 kV.
Images were recorded under low dose conditions (a total dose ∼25 e
−/Å2) with a 4000 SP 4 K slow-scan camera (Gatan, Pleasanton, CA) at
−390nm defocusandat a magnificationof 80,000. Thepixelsize(after
binning the images) was 3.75 Å at the specimen level and GRACE
software was used for semi-automated specimen selection and data
acquisition . In total, from each sample about 1500 images were
recorded and 15,000 single particle projections were selected
for image analysis. Single-particle analysis was performed with the
Groningen Image Processing software package (GRIP) on a PC cluster.
Selected single-particle projections were aligned by multi-reference
and reference-free alignment procedures [33,34]. Particles were then
subjected to multivariate statistical analysis followed by hierarchical
classification . Resolution was measured using Fourier-ring
correlation and the 3σ criterion .
Different PBS samples from Synechocystis were purified and ana-
lysed using the single particle EM technique. Purified PBS complexes
usually dissociate, unless high concentrations of phosphate are
present. However, high phosphate concentrations interfere with the
standard negative staining of samples for EM and often lead to noisy
backgrounds. A fixation procedure is therefore required in order to
remove phosphate before staining. For this reason, the purified com-
plexes were stabilised with glutaraldehyde on carbon-coated electron
microscope grids prior to negative staining, as described in more
detail in .
3.1. Purification and image analysis of core particles
The isolation of intact core complexes from native PBSs is not
feasible because AP trimers dissociate from the core before all rod PC
hexamers are detached. Therefore we used the Synechocystis CK
mutant, totally devoid of PC but still assembling intact core complexes
as judged by SDS-PAGE (Fig. 2) and by absorption spectroscopy (Ughy
and Ajlani, unpublished). Sucrose gradients of core preparations from
the CK mutant contained a unique turquoise band at a significantly
higher position than the dark-blue band found for wild-type PBSs.
Diluted samples of the turquoise fraction were immediately used to
prepare EM grids. Electron micrographs of CK particles show two
preferential orientations (top and side views) and a high homogeneity
of the sample (Fig. 3). Image analysis of a set of 10,882 negatively-
stained particles indicated that the cores were all intact particles
without any fragments.
Statistical analysis and classification gave different projection
views, presented in Fig. 4A–E. The side-view projections show that
the core complex is composed of three cylinders positioned in a
regular triangle with a maximal size of 210–220 Å, each of the three
sub-domains having a diameter of 110–117 Å (Fig. 4A–C). The X-ray
structure of a single AP trimer exhibits clear three-fold symmetry
. This symmetry is not observed in the individual AP core
cylinders in Fig. 4A, in contrast to EM projection maps of single rod
hexamers (Fig. 5Q). This indicates that the position of the AP trimers
associated with each cylinder may be translationally-shifted in a
direction perpendicular to the long axis of the cylinder. In Fig. 4B, C
and D the 2D map of the triangular core particle deviates
increasingly from its overall three-fold symmetry, indicating further
tilting of the whole particles on the carbon support film. Finally, it
should be remarked that the side-view maps of Fig. 4A–C clearly
show densities in the centre of the core cylinders. These additional
densities probably correspond to the linker proteins connecting the
trimers in each core cylinder.
Top views of the particles indicate that the three core cylinders
are in an overlap position, with only the outer portions of the two
Fig. 2. Polypeptide composition of PBS particles from wild type Synechocystis (WT),
from mutant strains CK, CB and CBFS, and for CBFS particles reconstituted with FNR
(REC). Polypeptide identities are given in the middle; masses of molecular markers are
indicated in kDa on the left.
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279
lower cylinders visible either side of the upper one (Fig. 4E). The
overall dimensions of the core complex, in this position, are about
155 Å wide by 220 Å long. From the staining pattern it appears that
the core cylinders of Synechocystis are each composed of four AP
discs of about 30 Å each. The staining profile also indicates a loose
interaction between AP trimers compared with the staining profile
within PC hexamers. It is obvious that the stain layer penetrates
more deeply between the AP trimers than in the PC hexamer, the
latter appearing as a single disc in all our projections (see Fig. 5).
The 2D EM map shows that the core has a strong two-fold rota-
tional symmetry in the top-view projection (Fig. 4E). This indicates
that the lower two cylinders are arranged in an anti-parallel
manner. The two-fold rotational symmetry is also strong in the
central parts of the complex where the upper cylinder contributes,
implying that the upper cylinder also has internal two-fold sym-
metry. The symmetry and the absence of tilted particles in the top
views suggest that the core particles are probably oriented on the
EM grids with their lower (membrane-facing) cylinders towards the
carbon film. Since the film surface is negatively charged after glow
discharge, interaction with arm2 of the LCM(known to be composed
of basic amino acids) might well be responsible for this specific
orientation of the core particles.
3.2. Analysis of PBS with shortened PC rods
Attempts to average images of wild-type PBSs yielded poor quality
maps (see Fig. 5P), probably due to the inherent instability and
flexibility of the rods. To avoid these problems we used the Syne-
chocystis CB mutant, in which the lack of two rod-linker proteins (LR
per rod (instead of three in the wild type). This mutant retained FNRL
at close to wild type levels . Diluted samples of the dark blue PBS
fractions from CB were immediately used to prepare EM grids. Two-
dimensional projection maps of negatively-stained particles are pre-
sented in Fig. 5A–D. The side-view map of Fig. 5A shows the attach-
ment of six shortened rods to the core; each of the shortened rods is
composed of a disc having the dimensions of a single PC hexamer. In
addition, some CB particles were missing one or more PC hexamers
(∼1% of the side views), due either to low stability or to the EM
specimen preparation procedure . Thus in Fig. 5B, all three
hexamers on the right-hand side of the complex are absent. The core
30) results in truncated PBSs containing only one PC hexamer
substructure in the CB PBS has a slightly better resolution than that of
CK (Fig. 4A), as alignment is improved for the less-symmetrical par-
ticles. The map shows two extra masses visible at the base of the
complex (green arrows; Fig. 5A, E and I), which may correspond to
less well-defined densities in the equivalent positions for the lower
resolution map in Fig. 4A. A possible candidate for these masses is
arm2 of the LCM, as discussed below.
The projection maps of the untilted top view(Fig. 5C) and a slightly
tilted top view (Fig. 5D) are 60 Å longer on each side than the core
complex (Fig. 4E) due to the presence of one PC hexamer on each side
of the core.
3.3. Relative position of PC rods on the core complexes
The side-views of the CB, CBFS and FNR-reconstituted CBFS
particles show six PC hexamers in side-view position (Fig. 5A, E–F, I–J
respectively). The staining pattern, however, is not equally strong for
all of them with some PC hexamers appearing much darker. In
general, the negative staining technique applied to large protein
complexes often does not lead to complete embedding in a homo-
geneous layer of stain from bottom to top of the structure. Instead,
the lower parts of a macromolecule, in close contact with the carbon
support film, are stronger stain-embedded than the upper parts. As a
result, lower parts of a structure appear darker in a 2D-projection
map. As exemplified in Fig. 5J, but also visible in Fig. 5A, E, F, I, K, we
can see that two out of six hexamers appear significantly darker, as
indicated by dark versus bright blue rectangles in Fig. 5J. This
indicates that the six different rods are not all in the same plane. In
the case of an ideal hemi-discoidal structure they would all have been
on the long axis through the centre of the core substructure. The
relative positions are difficult to quantify because in the top-view
maps the PC rods partly overlap. However, the outer ones at the base
of the structure do not overlap for more than half the surface and the
maps show that there are no clear displacements in the lower two
hexamers 1 and 6 (Fig. 5D; see Fig. 5J for numbering). Thus, most of
the displacement is in the two pairs of upper rods (2–5 and 3–4), i.e.
those which appear to be attached to the upper core cylinder.
3.4. Searching for the FNR
The position of FNR molecules was studied by analysing two new
types of PBS preparation: from the CBFS double mutant, in which
Fig. 4. Single particle analysis of negatively-stained CK particles. The three AP cylinders
typical of tri-cylindrical cores are clearly distinguishable in all side views. (A–C) Side-
view projections of the cores obtained after image analysis of 9500 particles, with a
resolution of 17 Å; (D) Tilted side-view projection generated by a class of 1382 single
projections, with a resolution of 21 Å; (E) Top-view projection of the cores obtained
from a data set of 5019 particles with a resolution of 13 Å. Red arrows indicate extra
densities at the lower left and upper right edges of the particle (see text). No symmetry
was imposed during or af ter image analysis.
Fig. 3. Electron micrograph of negatively-stained core particles isolated from the CK
mutant. Two preferential orientations (side and top views) are observed. The inset
shows some enlarged side views (upper row) and top views (lower row). The space bar
equals 1000 Å.
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279
the truncated CB PBS lacks FNR (Fig. 5E–H); and CBFS PBS recon-
stituted with FNR in vitro by the procedure described in 
(REC; Fig. 5I–M). Fig. 2 shows the polypeptide composition of the
PBSs used in this study. Both types of particle exhibit features
from the side- and top-view projection maps that are very similar
to those of the CB mutant (Fig. 5A–D). The side-view maps have
the same 13 Å resolution — note that the small densities at the
lower periphery in the side-views are again resolved (green
arrows, Fig. 5A, E, I).
A good indication of the position of FNR can be obtained by
difference mapping of particles with and without FNR in identical
top- or side-view positions. In such maps strong positive densities
will indicate the position(s) of FNR. The side-views are the best
maps for comparison because they show the least overlap of
components such as the PC hexamers. The resulting difference
maps (CB-minus-CBFS, REC-minus-CBFS) are shown in Fig. 5N, O for
(5I-minus-5E) and (5I-minus-5F), respectively. The strongest diffe-
rence is at a position between the two phycocyanin rods closest to
the membrane (red arrows, Fig. 5N, O). There are no significant
differences at the positions of the upper two PC hexamers.
4.1. Structural features of the phycobilisome
This EM analysis constitutes the first direct structural description
of how the core cylinders are associated, and extends the models of
the PBS core obtained following core dissociation experiments
[15,39]. In these models it was suggested that the core cylinders
would differentiate into type A and type B, so that two cylinders of
type A and one of type B form a three-cylinder core. Only the two
type-A cylinders, each composed of four different discs and a major
part of the LCM, would interact with the membrane and the reaction
centres within it since the bi-cylindrical core family is restricted to
two type-A cylinders . Our EM analysis of truncated PBSs of
Synechocystis 6803 has resulted in 2D maps to 13 Å resolution,
Fig. 5. Gallery of single particle EM images of Synechocystis phycobilisomes. No symmetry was imposed on any of the images. Green arrows indicate small densities at the lower side
of the AP core, which might play a role in membrane attachment. Light and dark blue rectangles illustrate positional differences of PC rods in side-views of truncated PBSs (see text).
Red arrows indicate the major differences in density between particles containing FNR and those without. (A–D) Averaged images of different PBS projections for CB, where the rods
are restricted to onlyone PC hexamer each and where FNRLis present; (A) Side-view map obtained aftera conditional sum of 4695 single particles, with a resolution of 14 Å; (B) Side-
view of a partially-disrupted particle, from a separate class of 934 individual particles; (C) Top-view of averaged PBSs from a data set of 1675 single particles at 18 Å resolution; (D)
Tilted top-view at 22 Å resolution of averaged PBSs from a data set of 1938 single particles. (E–H) Averaged images of CBFS particles, corresponding to CB without FNR. (E) Side-view
projection after image analysis of 2763 particles, at 13 Å resolution; (F) Slightly tilted side-view projection from 2150 particles, with a resolution of 16 Å; (G) Top-view projection after
processing of 1758 particles, at 13 Å resolution; (H) Tilted top-view from a data set of 1440 particles, at 16 Å resolution. (I–M) 2D-projections of the REC particle, obtained using CBFS
in which FNR was reconstituted in vitro. A data set of 6619 single side-view projections of REC PBS was collected; (I) represents the side view, at 13 Å resolution, while (J–K) are
slightly-tilted side-view projections at 15 and 20 Å resolution, respectively. A separate data of 6607 single particles was analysed in order to obtain top views of FNR-reconstituted
PBS: (L) Top-view projection at 16 Å resolution; (M) slightly-tilted top-view projection at 16 Å resolution. (N) Difference map (I) minus (E); (O) difference map (I) minus (F). (P)
Average of 68 4 side-view projections of the wild-type PBS; (Q) Sum of the PBS rod fragments in top view.
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279
providing new details of the overall structure and association of
core cylinders and PC rods. The projection map of core particles in
the top view shows how the four AP discs are arranged in the core
substructure. Our data indicate that the two lower cylinders are
arranged anti-parallel and that the upper cylinder also exhibits an
internal two-fold symmetry (see Fig. 1).
It has been shown by site-directed mutagenesis that the PB-loop
domain of the LCMdoes not play a role in PBS anchoring and should
thereforebe at the outer side of thebasal(type-A)cylinders of thecore
. Small densities at the top right and bottom left of the core top-
view in Fig. 4E (indicated by red arrows) could be the positions of the
PB-loops of the two LCMpolypeptides present in the core.
The fact that the top-view 2D projections of the cores replicate the
two-fold symmetry of the 3D model indicates that the particles are
oriented on the grids with their lower cylinders towards the
negatively charged carbon film. We propose that the novel densities,
revealed at the bottom of the two lower cylinders in the side-view
maps (green arrows, Fig. 5A, E, I), correspond to the arm2 domain of
the LCM linker. This domain has been proposed to play a role in
anchoring of the PBS to the thylakoid membrane . Since arm2 of
the LCMis known to be composed principally of basic amino acids,
electrostatic interaction between PBS and the photosystems might
occur in vivo since the presence of phospholipids and sulpholipids
gives a partial negative charge to the membrane surface. Six nega-
tively charged lipids are found exclusively at the cytoplasmic side of
the PSII dimer , which may play a role in PBS anchoring. On the
other hand, a PSI trimer contains less negatively charged lipids that
are buried between the monomers or under the peripheral subunits
. Thus electrostatic interaction might occur between PSII and the
PBS. Moreover, the overall symmetryand dimension of the core match
those of the PSII dimer, while they do not match those of PSI.
Furthermore, the PSII cytoplasmic surface is flat and could accom-
modate a PBS on its top. It would be difficult to imagine anything
similar for PSI cytoplasmic surface, which contains peripheral
subunits (see also last paragraph).
The top view image of the cores revealed a rotational two-fold
symmetry that confirms the antiparallel model for the basal cylinders
proposed by Glazer for the bi-cylindrical cores of Synechococcus .
For the bi-cylindrical core, this symmetry can be obtained whatever
the orientation of AP trimers within the cylinder is. The two-fold
rotational symmetry found in our tri-cylindrical samples implies that
at least in the upper cylinder, two types of interaction exist between
the four AP trimers, i.e. a face-to-faceinteraction between the T8and T
discs and a face-to-back one between the two central T discs (Fig. 1).
T8 and Talso constitute half of the basal cylinder and their interaction
must be similar to that in the upper cylinder. We propose that the
basal cylinders have a similar disc organisation except for the
replacement of T8–T trimers by B8–M trimers. This organisation is
similar to that found in the rods, where PC trimers are known to
aggregate face-to-face to form a tight hexamer that is further
assembled face-to-back into rods by the rod linkers.
During image analysis of the WT PBS sample, we observed an
abundance of smaller particles at the level of the micrograph. After
separate image analysis, an average projection of these smaller
particles is shown in Fig. 5Q. This view, which could represent indi-
vidual PC hexamers, shows a central mass composed of three small
protein densities. Three similar small densities are visible in the face
view of the upper core cylinder (Fig. 4 A, B), while they are not seen in
the lower two ones. This may indicate that the lower cylinders do not
make the same angle as the upper one in respect to a hypothetical axis
along the centre of the three cylinders. Alternatively there may be
different linkers present in the lower two AP cylinders.
The WT PBS is not only a large structure but appears to be rather
flexible as well, as averaged projection maps appear fuzzy,
particularly towards the ends of the rods (Fig. 5P). In contrast, the
maps of rod-truncated PBSs show well-preserved details, up to
about 13–14 Å in the best maps (Fig. 5A, E, I). The negative stain
distribution in the averaged side views indicates that not all the rods
have the same position where they bind to the core. On the other
hand, the top view does not show any substantial displacements in
the lower two hexamers 1 and 6 (Fig. 5D; numbering in Fig. 5J).
Rods 2 and 4, coloured dark blue in Fig. 5J, appear to be displaced.
Because the core structure exhibits two-fold symmetry, the position
of the six rods is related by the same two-fold rotational symmetry.
This allows us to propose a consistent model with displacements as
indicated in Fig. 6. Rods 2 and 3 are shifted outward in opposite
directions, but because rods 5 and 4 are related by symmetry then
these must be at the same off-axis position as rods 2 and 3, res-
pectively. Unfortunately, the top views of these particles do not give
further clues about the displacements of the rods, mainly because
they overlap with the core structure.
4.2. Localising the FNR
In our attempts to assign the location of FNR we examined
truncated PBSs for three different types of particle: (1) from the CB
mutant, which contains FNRL; (2) from the FNRL-lacking CBFS mutant;
and (3) CBFS PBS with reconstituted FNR (called REC). The projection
Fig. 6. Schematic model for the interaction of PBSs with PSII rows in Synechocystis. Side-
view projection of PBS containing only one PC hexamer per rod (core inpink and rods in
blue). Light green bars indicate small densities at the lower side of the core, which
might play a role in PBS membrane association via PSII subunits (note alignment with
dimer in C). (B) Top-viewof the Synechocystis core indicates that it matches the width of
the cyanobacterial PSII dimer (C) and double dimer (D). PSII dimer is outlined in dark
green. (E) Schematic arrangement of a single truncated PBS over a single PSII dimer,
seen in top view from the stromal side of the membrane. Numbers 1 to 6 (in blue)
indicate positions of the corresponding PC hexamers from (A). (F) Final model, viewed
from the stromal side of the membrane, indicates the most likely arrangement of WT
PBSs on PSII rows. Numbers 1-1-1 to 6-6-6 indicate positions of the three PC discks
within each of the six rods (in blue).
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279
maps show similar resolution for CB, CBFS and REC samples, so that
the small densities at the lower periphery of the side-views, assigned
to arm2 of the LCM, have been resolved in all three particles. By
subtracting the projections in which FNR is not present from those
where it is, difference maps were generated to see possible positions
of FNR. The best maps were produced by comparing the REC particle
with the CBFS PBS. A positive density is revealed at two symmetry-
related positions (Fig. 5N, O), as marked by red arrows. It suggests the
position of at least two FNR molecules. This position is at the interface
of the two lower PC rods, while there is no difference at the position of
the upper rods. The fact that we see the difference in Fig. 5N,O at two
symmetry-related positions, without having imposed any type of
symmetryon images during or after analysis, is good evidence in favor
of our FNR assignment.
4.3. Arrangement on photosystem II rows
About 20 years ago freeze-fracture EM convincingly revealed that
EF particles (PSII dimers) in PBS-containing organisms could be either
randomly distributed (in high light or state 2) or organised into rows
(in low light or state 1). The spacing between the EF particles in the
row was about 120 Å [42,43]. The periodicity of the particles in a row
is compatiblewiththewidthof a PBScore, whilethedistancebetween
the rows is proportional to the length of the PBS. In Synechocystis
thylakoids adapted to state 1, the distance between the rows is 500 Å
in the wild type, 230 Å in a rod-less mutant and 160 Å in a PBS-less
mutant . Similar distances between PSII were obtained by single
particle analysis based on negatively-stained specimens of solubilised
thylakoids, by measuring centre-to-centre distances between dimers
in the PSII double dimer . A periodicity of 122 Å and an inter-row
distance of 167 Å were obtained by Folea et al. .
Our PBS top-view projections indicate that the core is about 155 Å
in width (by 220 Å long), a similar but slightly larger width than
dimeric PSII (140 Å by 200 Å) . This is of interest because it would
mean that a 1:1 matching of PBSs on the top of PSII rows is only
possible when the PBSs make a lateral angle of about 17° with the PSII
row, as indicated in our model. The model shows how the two-fold
symmetrical PBSs would fit on top of rows of dimeric PSII, and refines
previous models for the relative position and attachment of PBS over
PSII rows. In our model the PBS is in contact with its neighbours on
adjacentPSIImoleculesin a row, inparticularvia thedisplacedPCrods
2 and 5, which might redistribute excitation energy between these
giant extra-membranous antennae, thus optimising light harvesting
The model presented above represents the best fit of existing 2D
electron projection data, but it is still based partly on indirect
observations. Up to now, no PSII complex with an associated PBS
has ever been characterised by EM and attempts to purify such
complexes give inconsistent results [47–49]. The exact position of
the PBS on PSII thus remains to be determined. The instability of the
PSII–PBS complex is a major drawback for its characterisation. One
way to improve stability would be to construct specific cysteine
mutants within PSII and PBS subunits. They could then be cross-
linked via disulphide bridges to give a megacomplex with higher
We thank B. Lagoutte for the generous gift of recombinant FNRL, B.
Robert for his support and A. Pascal for critical reading of the
manuscript. G. Oostergetel, W. Keegstra and J.-M. Verbavatz are
acknowledged for invaluable help with electron microscopy and
image processing. This project is part of the Dutch research
programme “From Molecule to Cell” funded by the Netherlands
Organization for Scientific Research (NWO) and the Foundation for
Earth and Life Sciences (ALW). Work in GA's lab was financially
supported by the CNRS, the CEA, and the research programme ANR-
This paper is dedicated to Claudie Vernotte.
 A.N. Glazer, Light guides. Directional energy transfer in a photosynthetic antenna,
J. Biol. Chem. 264 (1989) 1–4.
 M.H. Yu, A.N. Glazer, Cyanobacterial phycobilisomes. Role of the linker polypep-
tides in the assembly of phycocyanin, J. Biol. Chem. 257 (1982) 3429–3433.
 T. Schirmer, R. Huber, M. Schneider, W. Bode, M. Miller, M.L. Hackert, Crystal
structure analysis and refinement at 2.5 Å of hexameric C-phycocyanin from
the cyanobacterium Agmenellum quadruplicatum, J. Mol. Biol. 188 (1986)
 J.Y. Liu, T. Jiang, J.P. Zhang, D.C. Liang, Crystal structure of allophycocyanin from red
algae Porphyra yezoensis at 2.2-A resolution, J. Biol. Chem. 274 (1999)
 J. Nield, P.J. Rizkallah, J. Barber, N.E. Chayen, The 1.45 Å three-dimensional
structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus
elongatus, J. Struct. Biol. 141 (2003) 149–155.
 W.A. Sidler, Phycobilisome and phycobiliprotein structures, In: DA Bryant (Ed.),
The Molecular Biology of Cyanobacteria, Kluwer Acad. Pub., Dordrecht, 1994,
 E. Gantt, C.A. Lipschultz, Phycobilisomes of Porphyridium cruentum. I. Isolation,
J. Cell Biol. 54 (1972) 313–324.
 G. Guglielmi, G. Cohen-Bazire, D.A. Bryant, The structure of Gleobacter violaceus
and its phycobilisome, Arch. Microbiol. 129 (1981) 181–189.
 D.W. Krogmann, B. Perez-Gomez, E.B. Gutierrez-Cirlos, A. Chagolla-Lopez, L.
Gonzalez de la Vara, C. Gomez-Lojero, The presence of multidomain linkers
determines the bundle-shape structure of the phycobilisome of the cyanobacter-
ium Gloeobacter violaceus PCC 7421, Photosynth. Res. 93 (2007) 27–43.
 G. Yamanaka, A.N. Glazer, R.C. Williams, Molecular architecture of a light-
harvesting antenna. Comparison of wild type and mutant Synechococcus 6301
phycobilisomes, J. Biol. Chem. 255 (1980) 11104–11110.
 D.A. Bryant, R. de Lorimier, G. Guglielmi, S.E. Stevens Jr., Structural and
compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002.
Analyses of the wild-type strain and a phycocyanin-less mutant constructed by
interposon mutagenesis, Arch. Microbiol. 153 (1990) 550–560.
 R.C. Williams, J.C. Gingrich, A.N. Glazer, Cyanobacterial phycobilisomes. Particles
from Synechocystis 6701 and two pigment mutants, J. Cell Biol. 85 (1980) 558–566.
 K. Elmorjani, J.-C. Thomas, P. Sebban, Phycobilisomes of wild type and pigment
mutants of the cyanobacterium Synechocystis PCC 6803, Arch. Microbiol. 146
 D. Redecker, W. Wehrmeyer, W. Reuter, Core substructure of the hemiellipsoidal
phycobilisome from the red alga Porphyridium cruentum, Eur. J. Cell Biol. 62 (1993)
 A. Ducret, S.A. Muller, K.N. Goldie, A. Hefti, W.A. Sidler, H. Zuber, A. Engel,
Reconstitution, characterisation and mass analysis of the pentacylindrical
allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120,
J. Mol. Biol. 278 (1998) 369–388.
 M. Glauser, D.A. Bryant, G. Frank, E. Wehrli, S.S. Rusconi, W. Sidler, H. Zuber,
Phycobilisome structure in the cyanobacteria Mastigocladus laminosus and
Anabaena sp. PCC 7120, Eur. J. Biochem. 205 (1992) 907–915.
 D.J. Lundell, A.N. Glazer, Molecular architecture of a light-harvesting antenna.
Core substructure in Synechococcus 6301 phycobilisomes: two new allophyco-
cyanin and allophycocyanin B complexes, J. Biol. Chem. 258 (1983) 902–908.
 D.A. Bryant, Genetic analysis of phycobilisome biosynthesis, assembly, structure,
and function in the cyanobacterium Synecococcus sp PCC 7002, In: SE Stevens Jr.,
DA Bryant (Eds.), Light-Energy Transduction in Photosynthesis: Higher Plants and
Bacterial Models, American Society of Plant Physiologists, Rockville, 1988,
 V.Capuano, A.Braux,N. Tandeau de Marsac, J. Houmard,The“anchor polypeptide”
of cyanobacterial phycobilisomes. Molecular characterization of the Synecho-
coccus sp. PCC 6301 apcE gene. J. Biol. Chem. 266 (1991) 7239–7247.
 G. Ajlani, C. Vernotte, Deletion of the PB-loop in the LCMsubunit does not affect
phycobilisome assembly or energy transfer functions in the cyanobacterium
Synechocystis sp. PCC6714, Eur. J. Biochem. 257 (1998) 154–159.
 N. Adir, Elucidation of the molecular structures of components of the phycobili-
some: reconstructing a giant, Photosynth. Res. 85 (2005) 15–32.
 R. MacColl, Cyanobacterial phycobilisomes, J. Struct. Biol 124 (1998) 311–334.
 L.K. Anderson, C.M. Toole, A model for early events in the assembly pathway of
cyanobacterial phycobilisomes, Mol. Microbiol. 30 (1998) 467–474.
 W.M. Schluchter, D.A. Bryant, Molecular characterization of ferredoxin-NADP+
oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of
Synechococcus sp. PCC 7002 and studies on the gene product, Biochemistry
31 (1992) 3092–3102.
 J.J. vanThor, O.W. Gruters, H.C. Matthijs, K.J. Hellingwerf, Localization and function
of ferredoxin:NADP(+) reductase bound to the phycobilisomes of Synechocystis,
EMBO J. 18 (1999) 4128–4136.
 C. Gomez-Lojero, B. Perez-Gomez, G. Shen, W.M. Schluchter, D.A. Bryant,
Interaction of ferredoxin:NADP+oxidoreductase with phycobilisomes and
phycobilisome substructures of the cyanobacterium Synechococcus sp. strain
PCC 7002, Biochemistry 42 (2003) 13800–13811.
 B. Ughy, G. Ajlani, Phycobilisome rod mutants in Synechocystis sp. strain PCC6803,
Microbiology 150 (2004) 4147–4156.
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279
 J.C. Thomas, B. Ughy, B. Lagoutte, G. Ajlani, A second isoform of the ferredoxin:
NADP oxidoreductase generated by an in-frame initiation of translation, Proc.
Natl. Acad. Sci. U. S. A. 103 (2006) 18368–18373.
 M.M. Allen, Simple conditions for growth of unicellular blue-green algae onplates.
J. Phycol. 4 (1968) 1–4.
 A.N. Glazer, Phycobilisomes, Methods Enzymol. 167 (1988) 304–312.
 G. Ajlani, C. Vernotte, L. DiMagno, R. Haselkorn, Phycobilisome core mutants of
Synechocystis PCC 6803, Biochim. Biophys. Acta 1231 (1995) 189–196.
 G.T. Oostergetel, W. Keegstra, A. Brisson, Automation of specimen selection and
data acquisition for protein electron crystallography, Ultramicroscopy 74 (1998)
 P. Penczek, M. Radermacher, J. Frank, Three-dimensional reconstruction of single
particles embedded in ice, Ultramicroscopy 40 (1992) 33–53.
 M. van Heel, B. Gowen, R. Matadeen, E.V. Orlova, R. Finn, T. Pape, D. Cohen, H.
Stark, R. Schmidt, M. Schatz, A. Patwardhan, Single-particle electron cryo-
microscopy: towards atomic resolution, Q. Rev. Biophys. 33 (2000) 307–369.
 M. van Heel, Similarity measures between images, Ultramicroscopy 21 (1987)
 A.N. Glazer, R.C. Williams, G. Yamanaka, H.K. Schachman, Characterization of
cyanobacterial phycobilisomes in zwitterionic detergents, Proc. Natl. Acad. Sci.
U S A. 76 (1979) 6162–6166.
 K. Brejc, R. Ficner, R. Huber, S. Steinbacher, Isolation, crystallization, crystal
structure analysis and refinement of allophycocyanin from the cyanobac-
terium Spirulina platensis at 2.3 Å resolution, J. Mol. Biol. 249 (1995)
 B. Kastner, N. Fischer, M.M. Golas, B. Sander, P. Dube, D. Boehringer, K. Hartmuth, J.
Deckert, F. Hauer, E. Wolf, H. Uchtenhagen, H. Urlaub, F. Herzog, J.M. Peters, D.
Poerschke, R. Luhrmann, H. Stark, GraFix: sample preparation for single-particle
electron cryomicroscopy, Nat. Methods 5 (2008) 53–55.
 L.K. Anderson, F.A. Eiserling, Asymmetrical core structure inphycobilisomes of the
cyanobacterium Synechocystis 6701, J. Mol. Biol. 191 (1986) 441–451.
 B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Lipids in photosystem II:
 P. Jordan, P. Fromme, O. Klukas, H.T. Witt, W. Saenger, N. Krau, Three-dimensional
structure of cyanobacterial photosystem I at 2.5 Å resolution, Nature 411 (2001)
 E. Mörchel, M. Schatz, Correlation of photosystem II complexes with exo-
Synechococcus sp. Planta 172 (1987) 145–154.
 C. Vernotte, C. Astier, J. Olive, State 1–state 2 adaptation in the cyanobacteria
Synechocystis PCC 6714 wild type and Synechocystis 6803 wild type and
phycocyanin-less mutant, Photosynth. Res. 26 (1990) 203–212.
 J. Olive, G. Ajlani, C. Astier, M. Recouvreur, C. Vernotte, Ultrastructure and light
adaptation of phycobilisome mutants of Synechocystis sp. PCC 6803. Biochim.
Biophys. Acta 1319 (1997) 275–282.
 I.M. Folea, P. Zhang, E.M. Aro, E.J. Boekema, Domain organization of photosystem II
in membranes of the cyanobacterium Synechocystis PCC6803 investigated by
electron microscopy, FEBS Lett. 582 (2008) 1749–1754.
 A.A. Arteni, M. Nowaczyk, J. Lax, R. Kouřil, M. Rögner, E.J. Boekema, Single particle
electron microscopy in combination with mass spectrometry to investigate novel
complexes of membrane proteins, J. Struct. Biol. 149 (2005) 325–331.
 J. Clement-Metral, E. Gantt, Isolation of oxygen-evolving phycobilisome-
photosystem II particles from Porphyridium cruentum, FEBS Lett. 156 (1983)
 T. Katoh, E. Gantt, Photosynthetic vesicles with bound phycobilisomes from
Anabaena variabilis, Biochim. Biophys. Acta 546 (1979) 383–393.
 C.W. Mullineaux, Phycobilisome-reaction centre interaction in cyanobacteria,
Photosynth. Res. 95 (2008) 175–182.
ofthylakoidsof the cyanobacterium
A.A. Arteni et al. / Biochimica et Biophysica Acta 1787 (2009) 272–279