University of Groningen
Imaging of organelles by electron microscopy reveals protein-protein interactions in
mitochondria and chloroplasts
Dudkina, Natalya V.; Kouril, Roman; Bultema, Jelle B.; Boekema, Egbert
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FEBS Letters
DOI:
10.1016/j.febslet.2010.03.027
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Dudkina, N. V., Kouril, R., Bultema, J. B., & Boekema, E. J. (2010). Imaging of organelles by electron
microscopy reveals protein-protein interactions in mitochondria and chloroplasts. FEBS Letters, 584(12),
2510-2515. DOI: 10.1016/j.febslet.2010.03.027
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FEBS Letters 584 (2010) 2510–2515
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Review
Imaging of organelles by electron microscopy reveals protein–protein interactions
in mitochondria and chloroplasts
Natalya V. Dudkina, Roman Kouřil, Jelle B. Bultema, Egbert J. Boekema *
Electron Microscopy Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
a r t i c l e
i n f o
Article history:
Received 26 February 2010
Accepted 15 March 2010
Available online 19 March 2010
Edited by Jan Rydström
Keywords:
Electron microscopy
Tomography
Single particle electron microscopy
Mitochondrion
Chloroplast
Supercomplex
a b s t r a c t
Ongoing progress in electron microscopy (EM) offers now an opening to visualize cells at the nanoscale by cryo-electron tomography (ET). Large protein complexes can be resolved at near-atomic resolution by single particle averaging. Some examples from mitochondria and chloroplasts illustrate
the possibilities with an emphasis on the membrane organization. Cryo-ET performed on nonchemically fixed, unstained, ice-embedded material can visualize specific large membrane protein
complexes. In combination with averaging methods, 3D structures were calculated of mitochondrial
ATP synthase at 6 nm resolution and of chloroplast photosystem II at 3.5 nm.
Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
A first step in comprehension of metabolic processes is an
understanding of protein–protein interactions within organelles
or even complete cells. This goes beyond the level of the visualization of a cell ultrastructure with its nucleus, mitochondria,
endoplasmatic reticulum and other organelles. By fluorescence
microscopy many conclusions can be drawn about subcellular
localizations, and nowadays there is a whole toolbox of fluorescent
proteins assessing protein location and function [1]. But to understand protein–protein interactions we need a toolbox beyond the
micrometer scale of light microscopy. Ongoing progress in electron
microscopy (EM) offers now an opening to visualize cells at the
nanoscale. The first attempts to understand processes, such as cargo transport through nuclear pore complexes in intact nuclei, can
be solved with samples that did not undergo extensive chemical
treatment, fixation and staining [2], in marked contrast to the ones
on which the first concepts of subcellular ultrastructures were
developed in the 1950s of the last century. In this contribution,
we will discuss two techniques from the electron microscopy toolbox, electron tomography and single particle averaging, which are
useful utensils in structural biology. We further give examples of
these techniques, dealing with organelles with a highly complex
* Corresponding author.
E-mail address: [email protected] (E.J. Boekema).
system of inner membranes, in which protein–protein interactions
are crucial for efficient energy conversion.
2. Techniques for study of subcellular structures and large
biomacromolecules
Electrons can be accelerated to produce beams with a much
shorter wavelength than of visible light. This allows EM to be performed at atomic resolution. Contrast in EM arises from elastic
scattering of electrons. For weakly scattering objects, such as biological material, phase contrast is the most important part of contrast [3]. The signal is proportional to the electron dose, but
unfortunately the electron beam also damages biological molecules because inelastically scattered electrons deposit their energy
in the specimen. Because radiation damage should be kept at a
minimum this results in noisy images, especially for biological objects. Therefore, image analysis techniques have been developed to
improve the signal recorded by averaging over many projections of
the same molecule. Single particle averaging is nowadays the most
popular way of improving the signal-to-noise ratio.
Another point is the fact that EM produces 2D projections of the
imaged 3D objects. The superposition of features along the electron
beam direction results in images that can be ambiguous and
deceptive. To retrieve 3D information of subcellular structures, tilting of specimens in the electron microscope is necessary. To image
3D objects electron tomography (ET) has been developed. In ET, the
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2010.03.027
N.V. Dudkina et al. / FEBS Letters 584 (2010) 2510–2515
third dimension is retrieved by combing dozens of images recorded
under different tilt angles.
2.1. Single particle averaging
Single particle averaging has become popular because it can
deal with randomly oriented single molecules, which is a great
advantage over methods that need protein crystals. By adding hundreds or, if possible, hundreds of thousands of projections the signal improves substantially and trustworthy electron density maps
are obtained. The method of single particle analysis consists of a
few dedicated steps (see [4–7] for reviews on single particle EM).
Because single particles have random orientations on the support
film, averaging is possible after an alignment step. The alignments
bring projections in equivalent positions by computing rotational
and translational shifts. After alignment, particles can be summed
and by increasing the number of summed projections the noise is
gradually fading away. Just summing of projections, however, is
meaningless because they usually arise from particles in different
orientations toward the plane. In order to deal with this, data sets
have to be treated with multivariate statistical analysis together
with automated classification [4,5] to separate different angular
projections before they can be averaged. The fact that single particles are randomly oriented in a thin layer of amorphous ice has also
led to the development of 3D reconstruction methods that take
advantage of this [8].
Close to atomic resolution has been achieved by single particle
averaging for some biomolecules bearing high symmetry, which
enables interpretation of electron density maps at the level of amino acids [9]. For a stable, non-symmetric particle like the ribosome
fitting of the polypeptide chain is yet possible, but without highest
accuracy [10]. Many large protein complexes are, however, somewhat flexible. This severely limits the possibilities to obtain subnanometer resolution [7], as obtained for the ribosome. On the
other hand, even lower resolution data of large multi-subunit complexes can be very useful, in particular if they can be combined
with high-resolution structures of components. Fitting of X-ray
data into EM data is useful if the EM data are better than 15 Å, because fitting yields hybrid structures which are about 5 times better than the resolution of the EM structure [11]. This means that it
is already possible to look at helix–helix interactions with EM 2D
maps and 3D volumes slightly better than 15 Å.
2.2. Electron tomography
Electron tomography (ET) can provide three-dimensional information on any object with a thickness up to about 0.5 lm and is
very suitable to study biological structures with nanometer resolution (see [12–14] for recent reviews). It can be applied on quickly
frozen samples and imaging of unstained material in an amorphous layer of ice makes cryo-ET remarkably versatile, allowing
the exploration of a large range of biological specimens, both in
an isolated state and in their cellular context. By itself, ET is not
an averaging technique, because it only reconstructs a 3D volume,
the tomogram, by a combination of dozens of projections taken at
different tilt angles. However, by extracting sub-volumes corresponding to specific cellular components such as membrane proteins that are present in multiple copies, 3D averaging can be
used to obtain density maps of selected macromolecular complexes at progressively higher resolution [12]. This brings ET in
the realm of single particle averaging and many of the tools used
in the latter technique can be applied in ET as well [6]. Sorting of
molecules, for instance, demonstrated the ability of image classification to successfully separate the different conformations of the
HIV-1 envelope glycoprotein spikes [15]. The newly emerging
methods that combine cryo-ET with 3D image classification and
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averaging yield 3D images of molecular machines in a native environment, in a variety of biochemically well-characterized states,
unconstrained by intermolecular contacts characteristic for a crystal [16].
In the next two sections we provide some examples from mitochondria and chloroplasts that illustrate the possibilities of single
particle averaging and electron tomography, with an emphasis
on the membrane organization.
3. Mitochondrial supercomplexes
The organization of cristae membranes within mitochondria is a
very relevant topic because it now appears that the complexes involved in the oxidative phosphorylation system (OXPHOS) influence the morphology of these membranes. Moreover, it has been
found that the OXPHOS complexes form supramolecular assemblies termed respiratory supercomplexes or respirasomes [17]. In
recent years, the structure of the most relevant supercomplexes
has been determined by single particle analysis at medium resolution, after membrane solubilization and protein purification. The
most detailed data are from a yeast supercomplex formed by dimeric complex III and two monomeric copies of complex IV [18].
Fitting X-ray data with EM maps resulted in a pseudo-atomic model, describing positions of subunits involved in electron transfer between complexes III and IV. After the discovery of supercomplexes
it was questioned if these structures could be detergent artifacts.
Single particle averaging indicates this is unlikely because they
show specific interactions. If these supercomplex structures would
be artifacts, single particle analysis should have failed to reveal fine
details. Nevertheless, the possible influence of detergent has to be
faced. One aspect is that upon solubilization transient binding subunits are easily lost. An analysis of 80 000 particles of the III2–IV2
supercomplex of yeast showed that only a couple of dozen cytochrome c proteins remained attached to this supercomplex. There
are also other components that could transiently bind to the OXPHOS supercomplexes. For instance, the 30 kDa ADP/ATP carrier
loosely binds to the III2–IV2 supercomplex, but its association is
sensitive to detergent levels [19], and has not yet been detected
in EM projection maps.
The possible loss of supercomplex components and the difficulties to study protein–protein interaction after solubilization
prompted us to focus more on ET. Previously, the cristae structure
was studied by conventional EM methods, including chemical fixation and sectioning. This led to the standard baffle model for cristae structure that later was clearly shown to be inaccurate [20].
Depending on source and conformational state, cristae can vary
from simple tubular structures to more complex lamellar structures merging with the inner boundary membrane through tubular
structures 28 nm in diameter [20]. The structural information provided by cryo-ET can offer a lot of insight how supercomplexes are
distributed over the cristae membranes. Because tilting of samples
in a microscope is restricted to about 60–70°, it means that a substantial part of the 3D space is not sampled. This missing information is reduced if a second tilt series is recorded after rotating the
sample by 90°. Thus dual-axis ET provides a way to see subcellular
structures at higher resolution [21]. We applied dual-axis cryo-ET
to intact, close-to spherical mitochondria from the alga Polytomella
[22]. A slice through the center of an electron tomogram is presented in Fig. 1A. The difference in thickness between the outer
membrane and inner membrane is clearly visible. In this particular
mitochondrion, cristae are abundant; the slice suggests that they
are separated, but surface rendering of the membranes indicates
that they are interconnected. Surface rendering is also a tool to
visualize the folding of the inner membrane (coloured brown and
yellow, Fig. 1B) and to get an impression of the protein
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N.V. Dudkina et al. / FEBS Letters 584 (2010) 2510–2515
Fig. 1. Cryo-electron tomography of a mitochondrion from Polytomella and segmentation of its membranes. (A) Slice through the center of an electron tomogram. (B)
Rendering of the membranes of the mitochondrion represented in (A). The outer- (blue) and the inner membranes with invaginations (yellow/brown) have been rendered in
the upper-left part of the tomogram; red asterisks mark equivalent positions. Some cristae are outlined in blue–green and positions of F1 ATP synthase headpieces are in pink.
The space bar equals 200 nm. Surface rendering was performed with IMOD software [33].
distribution. The cristae membranes of Polytomella are densely
packed with ATP synthase complexes, and the 400 kDa sized F1
headpieces can even be mapped by hand (pink spheres, Fig. 1B).
The ATP synthase complexes are not homogeneously distributed
over the cristae membranes. In this and many other tomograms
it appears that the larger surfaces often appear rather smooth,
whereas smaller, highly curved cristae contain a lot more ATP synthase complexes (Fig. 1B). It appears that a part of the ATP synthase
dimers is organized into rows (Fig. 2B). The supramolecular organization of dimeric ATP synthase in the cristae membranes was further investigated by averaging sub-volumes of tomograms [22].
This showed that the dimers are at 12 nm intervals (Fig. 2C). Within the dimers the monomers are not in the same plane, but are under an angle [23]. This induces a local curvature of the membrane
of about 70° [22], and as a consequence cristae areas where ATP
synthase is concentrated appear more curved (Fig. 2D). This probably also means that the smooth cristae with rather flat surfaces
house the other OXPHOS components. Cryo-ET, however, is not
yet able to resolve these components. Nevertheless, averaging of
sub-volumes of tomograms with ATP synthase dimers provided
details at 6 nm resolution. At this resolution, the main features
of monomeric ATP synthase, such as the conically shaped F1 headpiece (marked F1 in Fig. 3A), the membrane-bound Fo part (Fo), the
central stalk and stator (S) are clearly visible for the first time in an
intact mitochondrion. These features are in the right proportion
compared to previously obtained single particle EM data
(Fig. 3C). This demonstrates the capability of dual-axis electron
tomography to unravel details of proteins and their interactions
in complete organelles.
4. Chloroplast membranes and photosystem II supercomplexes
Chloroplasts play a central role in the green plant energy
metabolism. They enclose the thylakoid membrane, which forms
a complicated three-dimensional network. The thylakoid membrane partly folds into stacks, called grana, which are interconnected by single membranes, the stroma thylakoids [24]. Both
Fig. 2. (A) A close look at cristae within a mitochondrion. (B) A tubular cristae densely packed with ATP synthase complexes. A row of ATP synthase dimers, seen from the top,
is within the pink square. (C) An average of subtomographic volumes of ATP synthase oligomers in a top view, corresponding to the pink square in (B). (D) Surface rendering of
the cristae (blue–green) from frame (B) in the same orientation. Row-like distribution of ATP synthase F1 headpieces is shown in pink. Scale bars are 50 nm for frame (B), (D)
and 20 nm for frame (C).
N.V. Dudkina et al. / FEBS Letters 584 (2010) 2510–2515
Fig. 3. Comparison of electron tomography and single particle electron microscopy
applied to dimeric ATP synthase from Polytomella mitochondria. (A) Average of
tomographic sub-volumes of ATP synthase dimer seen from a side revealing the F1
headpiece, the Fo membrane part and the peripheral stalk (S) or stator. (B) Same
average, seen from the top. (C) Average from negatively stained side-views of
isolated dimeric ATP synthase. The scale bar is 10 nm. Data modified from [22].
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tions, many PSII core particles have four LHCII trimers attached
and the PSII antenna size is maximal; in high light the complex dissociates, and LHCII migrates to the periphery of the grana. The dissociation of PSII supercomplexes leads to the formation of
quenching sites in the PSII antenna. This regulation of PSII supercomplex assembly is part of a control cycle connecting short term
energy dissipation to long term regulation of PSII antenna size
upon acclimation [26]. The formation of supercomplexes is only
one aspect of the complex interaction of thylakoid proteins. They
also interact and can form small crystalline domains, depending
on light conditions [27].
We have studied small stacks of grana thylakoid membranes by
cryo-ET to study the 3D membrane organization in more detail
(Kouřil et al., unpublished data). Fig. 4 presents a tomogram of a
granal disc obtained from Spinacia chloroplasts, which consists of
four membrane layers. Surface rendering shows that it actually
consists of two pairs of folded membranes; the outer membranes
(depicted in brown) and inner membranes (green) are connected.
The slices through the tomogram have been leveled in such a
way that the outer membrane makes the upper (A) and bottom
(D) section whereas the inner membrane is seen in Fig. 4B and C.
PSII complexes were resolved in all four membrane layers (brown
and green spheres for the outer and inner layers, respectively
(Fig. 4). They can be easily picked out, because the PSII core part
protrudes about 5 nm at one side of the membrane, whereas the
surrounding LHCII trimers, which lack membrane-extending domains, have two flat surfaces.
The tomograms of the grana stacks were also used for 3D single
particle averaging, in a way as performed for dimeric ATP synthase
[22]. An average of the core part of PSII, obtained by processing of
500 sub-volumes of Spinacia thylakoid membranes shows details
at about 3.5 nm (Fig. 5C). Processing is worth wile for more than
just one reason because it helps to get information about the orientation of the membranes. Because PSII only protrudes at the internal side of the thylakoid membranes it gives a clue to the absolute
orientation of the membrane, which is useful in ongoing studies
about dynamic changes in the chloroplast upon changing light
conditions.
Recently, a 2D map at 12 Å resolution was obtained for the PSII
supercomplex (Fig. 5A), which allowed determining the location
and the orientation of the individual light-harvesting components
[28]. As for mitochondrial supercomplexes, a medium resolution
map is a good starting point for construction a higher resolution
pseudo-atomic model by fitting of high-resolution X-structures
(Fig. 5B). The model derived shows a dimeric core particle flanked
by four LHCII trimers plus 2 3 monomeric antenna proteins. The
core part, as computed from cryo-ET, shows the same twofold symmetrical structure (Fig. 5C).
5. Concluding remarks and prospects
the grana – and stroma thylakoids are densely packed with large
membrane protein complexes involved in the light reactions of
photosynthesis (see [25] for a review). The three largest complexes
are Photosystem I (PSI), Photosystem II (PSII) and the ATP synthase
complex, whereas the most abundant one is light-harvesting complex II (LHCII). PSII and LHCII are mainly confined in grana stacks,
whereas PSI and ATP synthase are restricted to the unstacked stromal thylakoids [25]. It is important to understand the structural
organization of these complexes in the thylakoid membranes because it is closely related to their function. There are many indirect
evidences that the organization of photosynthetic apparatus
undergoes dynamic changes under varying environmental conditions, e.g. light intensity [26]. Within the grana stacks PS II and
LHCII are arranged into supercomplexes. Under low light condi-
The application of single particle averaging and cryo-ET has
undergone an exponential growth over the last decade, enabled
by technological advances in methods and instrumentation, and
is starting to make a significant impact on our understanding of
the cellular world. While the attained results are already remarkable, especially ET remains an emerging technique with ample potential to be exploited. Current developments towards large-scale
automation, and integration with other imaging techniques hold
promise for a near future in which ET will extend its role as a pivotal tool in structural and cell biology [14]. Resolution may be
pushed towards 2 nm, close to the theoretical limit.
Close to atomic resolution has been achieved by single particle
averaging for some biomolecules bearing high symmetry. This offers hope that the same can be done for molecules lacking it, but
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N.V. Dudkina et al. / FEBS Letters 584 (2010) 2510–2515
Fig. 4. 3D membrane organization of the granal thylakoid and PSII complexes. (A–D) Surface views of a granal thylakoid disc, which consists of four membrane layers.
Tomography shows that they consists of two pairs of membranes; the outer membranes (depicted in brown) and inner membranes (green). The outer membrane makes the
upper (A) and bottom (D) layer of the thylakoid disc, the inner membrane forms two layers inside the thylakoid disc (B and C). PSII complexes were resolved in four
membrane layers and are depicted as brown and green spheres for the outer and inner layers, respectively (Kouřil et al., unpublished data). The space bar equals 100 nm.
Fig. 5. Comparison of single particle electron microscopy and electron tomography applied to dimeric photosystem II C2S2M2 supercomplexes from Arabidopsis and Spinacia
thylakoid membranes. (A) Projection map at about 12 Å from negatively stained particles, obtained by averaging about 14 000 particles from Arabidopsis PSII [28]. (B) Pseudoatomic model derived from the EM data of frame (A), showing a dimeric core particle flanked by four LHCII trimers plus 2 3 monomeric antenna proteins (modified from
[28]). (C) An average of the core part of PSII from electron tomography, obtained by processing of 500 sub-volumes of Spinacia thylakoid membranes. No symmetry was
imposed on this sum during or after the analysis (Kouřil et al., unpublished data). Space bar equals 100 Å.
it gives daunting estimates on the number of particles to be collected and processed [16]. Currently, to reach atomic resolution
for non-symmetric biomolcules by a conventional cryo-EM, about
1 000 000 particle projections are required [29]. According to Joachim Frank, one of the founding fathers of single particle averaging,
it is difficult to find software recipes for improving the resolution
of beyond what has been achieved, except for brute-force increasing data collection [16]. On the other hand, instrumental improvements are still possible and this will eventually lead to higher
resolution with smaller numbers of particles. Two examples are given here. The first has to do with recording of images. For decades,
images were recorded manually on film. When automation became
necessary, films were replaced by slow-scan charged-coupled device (CCD) cameras. CCD detectors are used mainly in an indirect
mode: energy deposited by the primary electron in a phosphor
or scintillator is imaged through either fiber optics or a lens onto
a CCD. The problem with indirect detection is that spatial resolution is reduced as light suffers from multiple scattering within
the phosphor or at the optical interfaces [30]. Recently, direct electron counters were developed. Direct electron technology ensures
a direct translation from electrons towards the image without
interference of an optically coupled device. This leads to improved
spatial resolution and superior image quality. The first commercial
camera was launched in 2009 (see http://www.fei.com and http://
www.nanotech-now.com/news.cgi?story_id=34033). The second
example is about the improvement of the contrast of cryo-images.
This is all important because the alignment step of individual projections will become more accurate when the signal improves.
N.V. Dudkina et al. / FEBS Letters 584 (2010) 2510–2515
Enhancement of contrast can be achieved by implementing a
Zernike phase plate. In this way the number of particles required
to attain near-atomic resolution can, in principle, be reduced by
10–100 fold for proteins between 100 kDa and 500 kDa [31]. First
experiments with Zernike phase plates have shown a tremendous
improvement in contrast [32], but application is not yet a standard
routine.
In conclusion, the EM toolbox offers techniques with increasing
impact to tackle a wide scale of issues about protein–protein interaction in supercomplexes, membranes and intact organelles.
Acknowledgements
This work was supported by the Netherlands Organization for
Scientific Research (NWO), Council for Chemical Sciences.
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