ARTICLE
Received 8 Jul 2015 | Accepted 23 Dec 2015 | Published 28 Jan 2016
Volta phase plate cryo-EM of the small protein
complex Prx3
Maryam Khoshouei1,*, Mazdak Radjainia2,3,*, Amy J. Phillips4, Juliet A. Gerrard2,5,6, Alok K. Mitra2,
Ju¨rgen M. Plitzko1, Wolfgang Baumeister1 & Radostin Danev1
Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers
of high-resolution structures since the arrival of direct electron detectors. However,
sub-nanometre resolution cryo-EM structures are rare compared with crystal structure
depositions, particularly for relatively small particles (o400 kDa). Here we demonstrate the
benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure
determination of 257 kDa human peroxiredoxin-3 dodecamers at 4.4 Å resolution. The
Volta phase plate improves the applicability of cryo-EM for small molecules and accelerates
structure determination.
DOI: 10.1038/ncomms10534 OPEN
1 Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. 2 School of Biological Sciences, University of
Auckland, Auckland 1042, New Zealand. 3 Department of Biochemistry and Molecular Biology, Monash University, Victoria, 3800 Melbourne, Australia.
4 Lincoln Agritech Limited, Lincoln 7640, New Zealand. 5 MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University, Wellington
6140, New Zealand. 6 School of Chemical Sciences, University of Auckland, 23 Symonds Street, Auckland 1010, New Zealand. * These authors contributed
equally to this work. Correspondence and requests for materials should be addressed to R.D. (email: danev@biochem.mpg.de).
NATURE COMMUNICATIONS | 7:10534 | DOI: 10.1038/ncomms10534 | www.nature.com/naturecommunications 1
C
ryo-electron microscopy (cryo-EM) has long held the
promise to deliver high-resolution structures of biological
assemblies without the need for crystallization1. Direct
electron detectors are proving pivotal in fulfilling this promise,
with the recent publications of many atomic or near-atomic
models derived from cryo-EM maps2. However, successes such as
those reported by Liao et al.3, Bai et al.4 and Bartesaghi et al.5
often represent well-behaved specimens that are exceptions
rather than the norm. The challenges in obtaining successful,
high-resolution cryo-EM images are manifold. Factors that
ultimately dictate the attainable resolution include particle size,
rigidity, sample purity, orientation coverage and ice thickness6.
Optimizing these parameters can be complex, prohibitively
time-consuming, and does not always lead to high-quality
maps. These potential issues severely limit a broader
applicability for single-particle cryo-EM.
The major impediment in achieving sub-nanometre resolution
cryo-EM reconstructions across the proteome is the intrinsically
low signal-to-noise ratio obtainable with such unstained
biological samples embedded in ice1. The recently proposed
Volta potential phase plate (VPP) addresses this long-recognized
problem of cryo-EM7,8 and has many advantages over previous
phase plate designs including durability and ease of use9. It is a
remarkably simple device, comprising a heated film of
continuous, amorphous carbon located at the back-focal plane
of the objective lens (Supplementary Fig. 1). The VPP introduces
a phase shift in the unscattered beam relative to scattered beams,
increasing the phase contrast, and thus facilitates the observation
of weak phase objects with markedly improved contrast.
While the use of VPP has already proven essential in
cryo-electron tomography studies to reveal structural details of
26S proteasomes in intact neurons10, its benefits to single-particle
analysis have not yet been demonstrated. We sought to
investigate the potential for structure determination using VPP
with single-particle analysis, specifically for solving the cryo-EM
structure of the 257-kDa, toroidal dodecamers of human
peroxiredoxin-3 (hPrx3; ref. 11) as an example for tackling
relatively ‘small’ protein complexes.
Peroxiredoxins (Prxs) are a ubiquitous class of peroxidases that
mediate the detoxification of reactive oxygen species or detection
of cellular stress, with a concomitant change of quaternary
organization12. hPrx3 is a human mitochondrial isoform that
forms dodecamers when the central cysteine is in its reduced
form. The dodecamers can further assemble into long
filaments upon acidification13. We recently obtained cryo-EM
reconstructions of hPrx3 filaments and observed a structured C
terminus11, which has not been previously solved in homologous
crystal structures, such as that of bovine Prx3 (ref. 14).
Determination of high-resolution structures for non-crystalline
hPrx3 samples can be laborious in practice as recently reviewed
by Cheng et al.6. This is owing to the following two reasons: first,
complexes with a molecular weight below 300 kDa exhibit very
poor contrast and are particularly susceptible to beam-induced
motion; and second, toroidal structures such as the hPrx3
dodecamers often assume preferred orientations in thin ice layers
and at low concentrations6. These issues can be overcome by
using thicker ice and increased protein concentration, but this
leads to complications in terms of further reduced contrast and
difficulties in particle detection6.
In this study, we report the 4.4-Å resolution structure of
257 kDa hPrx3, which paves the way for routinely determining
relatively small complexes at near-atomic resolution by
VPP-enabled phase contrast cryo-EM.
Results
3D reconstruction from a manually collected dataset. We
sought to elucidate the conformation that hPrx3 assumes in
solution, without the potential for structural changes that may
occur during assembly to higher-order structures or crystal
packing. Frozen-hydrated specimens of hPrx3 were prepared by
relaxing the blot force of the automatic plunging device to
Nyquist frequency = 3.5 Å
a b c
d e
Figure 1 | Near-atomic resolution reconstruction of hPrx3. (a) Electron micrograph of frozen-hydrated hPrx3 dodecamers taken without a phase plate at
2.4 mm underfocus. In thick ice, toroids are only readily discernible at high defocus (scale bar, 100 nm). (b) In-focus electron micrograph of hPrx3 particles.
VPP images of hPrx3 are high in contrast even in-focus and before motion correction, enabling robust automated particle selection of all views (scale bar,
100 nm). (c) Power spectrum of electron micrograph featuring continuous signal without contrast transfer function (CTF) oscillations across the frequency
spectrum. (d) Four representative class averages of hPrx3 featuring random orientations promoted by thick ice (scale bar, 10 nm). (e) Isosurface
representation of the reconstructed 3D density map with bovine Prx3 dimer structure docked (PDB 1ZYE (ref. 14)).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10534
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increase ice thickness. On the first attempt, hPrx3 particles were
visualized with high contrast, dense distribution and random
orientations (Supplementary Fig. 2). Forty-four in-focus images
were recorded manually in movie mode, from which a 7.3-Å
single-particle cryo-EM reconstruction was obtained, resolving
secondary structure elements (Supplementary Fig. 3).
Already, at this level of resolution, a-helices are clearly
discernible and reveal a volume of density that can be assigned
to a folded C terminus, similar to that seen in the reconstructions
of hPrx3 filaments as well as other Prx isoforms15
(Supplementary Fig. 3). This finding highlights the strengths of
using VPP for single-particle analysis, which include greatly
reduced requirements for optimization of sample preparation,
and structure determination to sub-nanometre resolution with a
relatively small number of images. Therefore, one can efficiently
delineate the structure of a complex in solution to complement
structural information obtained by X-ray crystallography. The
ability to induce and visualize alternate orientations of hPrx3
using thicker ice is particularly noteworthy, which would have
otherwise restricted us to a low-resolution random conical tilt
reconstruction. In fact, we attempted to reconstruct hPrx3
without phase plates using a grid from the same batch that
yielded the 7.3-Å phase plate structure. However, we were not
able to obtain a reconstruction that is consistent with the
structure of hPrx3, let alone well resolved, despite investing more
effort in particle picking and processing. Both data sets were
made available for readers that would like to compare the
respective raw data.
3D reconstruction from an automatically collected dataset.
Since our first VPP data set of hPrx3 featured a significant
amount of drift due to instability of the support film, we recorded
another data set using a different grid type to see whether resolution
can be improved. Indeed, with the new data set, we were
able to obtain a reconstruction of hPrxs at a resolution of 4.4 Å
from 8,500 particles (Fig. 1). This boost in resolution markedly
improved the quality of the map allowing for more precise
interpretation of secondary structure motifs, including the
C-terminal alpha helix, which was not fully resolved in the 7.3-Å
map (Fig. 2).
This result clearly shows that VPP imaging is not limited to
intermediate resolutions, which has been observed for other
phase plate designs16 and can, in the case of hPrx3, even deliver a
near-atomic resolution. It should also be noted that the second
map was sharpened with a smaller B-factor of À 137. This value
suggests a less steep fall-off of high-resolution information,
indicating that even higher resolution reconstructions may be
possible using VPP.
Discussion
In close-to-focus images taken with a VPP, contrast transfer is
maximal and continuous for low-to-high spatial frequencies. The
principles underlying this phenomenon are similar to light
microscopy where phase plates have been commonplace since the
1940s (ref. 17), and which have been finally realized in a practical
solution for transmission electron microscopes through the VPP.
To have maximum contrast and obtain sub-8 Å resolution
surpasses current alternatives for many applications8. Without
phase plates, microscopists can primarily defocus the objective
lens—a deliberate aberration of the microscope optics—to
increase the contrast of weak phase objects. This is not an issue
with ideal cryo-EM specimens, for which molecular weight and
isotropic coverage in thin ice are not limiting factors. In many
cases, however, a useful compromise between contrast and
resolution cannot be obtained, particularly when contrast is
extremely weak and very high defocus levels are needed to
compensate6. Defocusing introduces oscillations in the transfer of
contrast as a function of spatial frequency, which together with
ba
C-terminus′
C-terminusα6
α6′
1.2
1
0.8
Fouriershellcorrelation
0.6
0.4
0.2
0
0 0.05 0.1 0.15
Resolution (Å–1
)
1/6.5 Å
1/4.4 Å
0.2 0.25 0.3
Figure 2 | 3D density map with docked X-ray structures. (a) The VPP reconstruction of hPrx3 is consistent with the crystal structure of hPrx2
(PDB 1QMV (ref. 15)), where the dimer features folded C terminus (red; top). The C-terminal region is not present in the crystal structure of the bovine
homologue of Prx3 (PDB 1ZYE (ref. 14)) and disappears in our EM map at lower thresholds (middle), suggesting only partial occupancy of the folded C
terminus. The obtained resolution is sufficient for resolving individual beta-strands in the density map (bottom). (b) Fourier-shell correlation indicates a
resolution of 4.4 Å based on the ‘gold-standard’ Fourier shell correlation (FSC) ¼ 0.143 criterion.
Table 1 | Data collection parameters and refinement
statistics.
Data collection parameters
Pixel size (Å) 1.74
Defocus (nm) 20
Voltage (kV) 300
Electron dose (eÀ per Å2) 20
Refinement and modelling statistics
Particles for refinement 8,500
Resolution (Å) 4.4
Map sharpening B-factor (Å2) À 137
Cross-correlation coefficient for PDB 1ZYE 0.85
Cross-correlation coefficient for PDB 1QMV (dimer) 0.84
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the effect of the envelope function attenuate high-resolution
components in the image6.
Despite the B18% signal loss with the VPP9, it is still possible
to obtain near-atomic resolution structures even for a relatively
small complex such as hPrx3. This contrasts with previous
efforts using alternative phase plate designs where signal decay at
high resolution, reflected in very high B-factors, impedes
high-resolution structure determination16.
We believe that VPP single-particle analysis will add great
value in two ways. First, it can act in a complementary role to
high-resolution structure determination techniques such as X-ray
crystallography, defocus contrast-based single-particle analysis or
nuclear magnetic resonance, and will be a powerful alternative to
negative staining techniques or small-angle X-ray scattering.
Second, the example of hPrx3 also heralds a role for phase plate
technology as a high-resolution technique that can be used stand
alone.
Given several prominent successes in high-resolution
structures of small structures without phase plates4,18, it is
reasonable to assume that complexes such as hPrx3 could also be
solved to high-resolution by a defocus-based approach. However,
because of the effort required, this is unlikely to become the norm
in the short term.
At the time of writing of this manuscript, according to the
Electron Microscopy Data Bank (URL: http://www.emdatabank.
org), single-particle cryo-EM has been used to produce 237 nearatomic
resolution structures with around 136 structures being
either for systems related to ribosomes or highly symmetric
viruses. The number of cryo-EM structures solved to 8 Å or
higher resolution in 2014 was 185. Of the B3,000 total released
EM maps, B70% have a resolution of 10 Å or lower. Comparison
of these numbers with X-ray crystallography depositions5 reveals
the significant potential to grow the cryo-EM output and increase
the number of laboratories participating in such an endeavour.
We believe that by using VPP technology, the potential for the
study of biological systems that challenge cryo-EM can be
significantly increased, thereby enabling visualization of even
smaller protein complexes, highly flexible proteins or systems
requiring contrast-reducing solutes. VPP single-particle analysis
will enable higher throughput by increasing the success of
cryo-EM sessions, which may have previously failed due to ice
thickness, poor sample purity or insufficient particle numbers.
At the same time, VPP also has the potential to completely
replace negative staining, which, despite obvious artefacts in
many instances, is still widely used for difficult samples and in the
initial screening stages of promising cryo-EM projects that may
reach near-atomic resolution.
Taken together, the VPP with its tolerance for recalcitrant
specimens may follow the success of the Zernike phase plate in
light microscopy and find widespread adoption for routine use in
the determination of sub-nanometre resolution structures
through single-particle analysis.
Methods
Protein expression and purification. Prx3 was purified as described previously11.
Briefly, protein expression was performed in Rosetta (DE3) cells (Novagen), which
were lysed in binding buffer (20 mM HEPES, 150 mM NaCl and 10 mM imidazole,
pH 8.0) using a cell press (Microfluidics M110-P lab homogenizer). Soluble
fraction were subjected to affinity purification using a 5-ml HisTrap FF column
(GE Healthcare and successively subjected to gel filtration using a HiLoad 16/60
Superdex 200 column (GE Healthcare) using imidazole-free gel filtration buffer
(20 mM HEPES and 150 mM NaCl, pH 8.0). His-Tag cleavage was mediated
by recombinant TEV protease in the presence of 2 mM beta-mercaptoethanol
followed by removal of cleaved His-tags and uncleaved protein using a 1-ml
HisTrap column (GE Healthcare).
Preparation of vitrified specimen. For the first data set, frozen-hydrated
specimens were prepared on plasma-cleaned holey EM grids (Quantifoil R2/1)
using a Vitrobot Mark III (FEI) 4 s blotting time, 85% humidity and À 1 mm
blotting offset. For the second data set, frozen-hydrated specimens were prepared
on plasma-cleaned holey EM grids (Quantifoil R1.2/1.3) using a Vitrobot Mark III
(FEI) 4 s blotting time, 85% humidity and À 1 mm blotting offset.
EM and image processing. For the first data set, 44 movies were manually
collected on a Titan Krios electron microscope (FEI) operated at 300 kV and
equipped with a K2 Summit direct detector, a quantum energy filter (Gatan)
and an FEI phase plate (FEI). Movies comprising 10 frames and a total dose
of 20 eÀ per Å2 were recorded on a K2 Summit direct detection camera (Gatan)
at a calibrated magnification of 37,000 corresponding to a magnified pixel size
of 1.35 Å.
The recorded movies showed obvious signs of drift and were subjected to
combined global19 and local movement correction by optical flow analysis20.
One micrograph was subjected to e2boxer in EMAN2 (ref. 21) for initial particle
picking. The particles were extracted in Relion22 using a box size of 152 pixels.
This small data set comprising 500 particles was used for reference-free
two-dimensional (2D) classification in Relion with 10 classes. Of those 10 classes,
three classes were deemed suitable to represent the three dominant views—top view,
side view and tilted view. These three classes served as templates for automatic
particle picking in Relion yielding 30,000 particles. 2D classification of the full data
set was used to remove particles that were either false positives or belonged to classes
featuring touching particles resulting in a data set of 10,000 particles. The reduced
data set was subjected to three-dimensional (3D) classification using the crystal
structure of bovine Prx3 dodecamers as initial model after low-pass filtering it to
60Å, which produced a featureless toroid. 3D classification was used to group the
images into three 3D classes imposing D6 symmetry. 7,000 particles were assigned to
the most prominent 3D class, which was further processed with auto-refinement in
Relion. This yielded a map, which was sharpened in Relion using a negative B-factor
of 800. For resolution determination, the so-called ‘gold-standard’ Fourier shell
correlation (FSC) ¼ 0.143 criterion was used. 3D visualizations of the map and rigidbody
dockings were carried out in UCSF Chimera23.
For the second data set, 700 micrographs were recorded on a Titan Krios
electron microscope operated at 300 kV and equipped with a K2 Summit direct
detector, a quantum energy filter (Gatan) and an FEI phase plate (FEI). Automatic
data collection was carried out with SerialEM24 software. Images were recorded in
movie mode comprising 15 frames with a total dose of 20 eÀ per Å2 at a
magnification of 28,700 and a pixel size of 1.74 Å. The frames were aligned as
described by Li et al.19. Particles (354,000) were semi-automatically picked using
EMAN2 (ref. 21) e2boxer software and extracted in Relion 1.3 with a box size of
152. False positives were removed by three rounds of 2D classification followed by
four rounds of 3D classification. 8,580 particles from the best looking 3D class were
subjected to 3D auto-refinement in Relion. Particle-based beam-induced motion
correction was performed as part of the movie processing. The produced map was
then sharpened in Relion with automatically estimated B-factor of À 137. The
data collection parameters and refinement statistics for the second data set are
summarized in Table 1.
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Acknowledgements
We thank Vahid Abrishami for help with motion correction of the initial data set. This
work was funded by Instruct (grant reference PID: 1300), part of the European Strategy
Forum on Research Infrastructures (ESFRI) in collaboration with the Instruct Image
Processing Center I2PC. A.K.M. and M.R. were supported by a grant from NZ Health
Research Council; M.R. acknowledges travel support from the School of Biological
Sciences, University of Auckland and the contribution of the New Zealand eScience
Infrastructure (NeSI) high-performance computing facilities at the University of Auckland,
and the staff at NeSI and Centre for eResearch.
Author contributions
M.K., M.R., J.M.P. and R.D. were responsible for the conception, design, execution and
interpretation of experiments. A.J.P. purified protein. All authors contributed to the
writing of the manuscript.
Additional information
Accession codes: The cryo-EM map of the 4.4-Å resolution reconstruction has been
deposited in the Electron Microscopy Data Bank (EMDB code: EMD-3233). In addition,
we provide access to the raw images of hPrx3 recorded with and without the use of VPP
(URL: http://dx.doi.org/10.6019/EMPIAR-10050).
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
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How to cite this article: Khoshouei, M. et al. Volta phase plate cryo-EM of the small
protein complex Prx3. Nat. Commun. 7:10534 doi: 10.1038/ncomms10534 (2016).
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