of 18
Article
Structure of
Anabaenaflos-aquae
gas vesicles
revealed by cryo-ET
Graphical abstract
Highlights
d
Gas vesicles (GVs) are formed by

3-nm corrugated protein
shells
d
Corrugation reverses at the cylinder midpoint, which may act
as an elongation center
d
The protein shell is primarily formed by the conserved major
structural protein GvpA
d
GvpC provides extra reinforcement by forming a helical spiral
around the GV cylinder
Authors
Przemys
1
aw Dutka,
Lauren AnnMetskas,RobertC.Hurt, ...,
Tsui-Fen Chou, Mikhail G. Shapiro,
Grant J. Jensen
Correspondence
mikhail@caltech.edu (M.G.S.),
grant_jensen@byu.edu (G.J.J.)
In brief
Dutka et al. used cryo-ET supported by
biochemical data and computational
modeling to reveal the conserved
structure of
Anabaena flos-aquae
gas
vesicles. The resulting model gives
insights into the distinctive mechanical
properties of gas vesicles and their
assembly.
Dutka et al., 2023, Structure
31
, 518–528
May 4, 2023
ª
2023 The Author(s). Published by Elsevier Ltd.
https://doi.org/10.1016/j.str.2023.03.011
ll
Article
Structure of
Anabaenaflos-aquae
gas vesicles revealed by cryo-ET
Przemys
1
aw Dutka,
1
,
2
Lauren Ann Metskas,
2
,
7
,
8
Robert C. Hurt,
2
Hossein Salahshoor,
3
Ting-Yu Wang,
2
,
4
Dina Malounda,
1
George J. Lu,
1
,
9
Tsui-Fen Chou,
2
,
4
Mikhail G. Shapiro,
1
,
5
,
*
and Grant J. Jensen
2
,
6
,
10
,
*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
3
Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA 91125, USA
4
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
5
Howard Hughes Medical Institute, Pasadena, CA 91125, USA
6
College of Physical and Mathematical Sciences, Brigham Young University, Provo, UT 84602, USA
7
Present address: Biological Sciences Department, Purdue University, West Lafayette, IN 47907, USA
8
Present address: Chemistry Department, Purdue University, West Lafayette, IN 47907, USA
9
Present address: Department of Bioengineering, Rice University, Houston, TX 77005, USA
10
Lead contact
*Correspondence:
mikhail@caltech.edu
(M.G.S.),
grant_jensen@byu.edu
(G.J.J.)
https://doi.org/10.1016/j.str.2023.03.011
SUMMARY
Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea
as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs
have led to their use as genetically encodable contrast agents for ultrasound and MRI. Currently, however,
the structure and assembly mechanism of GVs remain unknown. Here we employ cryoelectron tomography
to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament
changes polarity at the center of the GV cylinder, a site that may act as an elongation center. Subtomogram
averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a
b
sheet. The
accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement.
Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt
different diameters and shapes.
INTRODUCTION
A fundamental property of many living organisms is their ability to
move within their environment, with single-celled organisms
capable of swimming, swarming, and aligning with magnetic
fields. The molecular machines underlying many of these motility
functions have been characterized in detail.
1–3
However, the
structure underlying one of the oldest evolved forms of motility,
flotation, remains more mysterious. Some cyanobacteria, hetero-
trophic bacteria, and archaea regulate their buoyancy in aquatic
environments to access sunlight and nutrients using intracellular
flotation devices called gas vesicles (GVs).
4
,
5
These unique
protein nanostructures consist of a gas-filled compartment, typi-
cally

100nmindiameterand

500 nm in length, enclosed by
a

3-nm-thick protein shell (
Figure 1
A) that can withstand hun-
dreds of kilopascals of applied pressure.
6
,
7
The interior of the shell
is strongly hydrophobic, keeping out water while allowing gas
molecules to diffuse in and out on a sub-millisecond timescale.
4
,
5
In addition to their biological significance, GVs are a subject of
intense interest for biotechnology. Analogous to fluorescent pro-
teins, opsins, and CRISPR nucleases, GVs’ unusual biophysical
properties can be harnessed for other purposes. The gaseous
composition of GVs allows them to scatter ultrasound waves,
enabling their use as genetically encoded reporters and actua-
tors of cellular function deep in tissues.
8–14
Other applications
take advantage of GVs’ refractive index, gas permeability, and
susceptibility to magnetic fields.
15–17
GVs were discovered in the 19th century, but we still have
limited knowledge of their structure and assembly. GVs adopt
a cylindrical shape with conical caps (
Figure 1
A). Their compo-
nents are encoded in operons containing relatively few genes
(8–23+, depending on the species).
5
One of these genes en-
codes the main structural protein, GvpA, a small (

8-kDa), highly
hydrophobic protein that polymerizes to form the GV shell.
4
In
some species, the gene cluster contains a secondary structural
protein called GvpC, which binds to the exterior of the shell to
provide mechanical reinforcement.
18
The remaining genes
encode proteins whose functions are not well understood,
possibly including chaperones, assembly factors, and additional
minor shell constituents. GVs are nucleated as bicones that then
elongate into a cylindrical shape with low-pitch helical ribs,
5
,
19
but their detailed molecular structure is not known.
Here, we apply state-of-the-art cryoelectron tomography
(cryo-ET) and subtomogram averaging techniques to GVs from
518
Structure
31
, 518–528, May 4, 2023
ª
2023 The Author(s). Published by Elsevier Ltd.
This is an open access article under the CC BY license (
http://creativecommons.org/licenses/by/4.0/
).
ll
OPEN ACCESS
the cyanobacterium
Anabaena flos-aquae
(Ana). These GVs are
among the best studied by biophysicists
4
,
20
,
21
and the most
commonly used in biotechnology applications.
13
,
22
,
23
We show
that the Ana GV shell is formed by a continuous helical filament
of repeating GvpA subunits, giving rise to a corrugated cylindri-
cal structure with terminal cones that taper over a conserved
distance. Near the middle of the cylinder, the angle of corruga-
tion is inverted, suggesting a potential elongation center for GV
biosynthesis. The corrugated shell is externally reinforced by
circumferential rods of GvpC. Combining our cryo-ET data with
an atomic model of the homologous
Bacillus megaterium
(Mega) GvpA protein determined in a complementary study,
24
we build an integrative model of the Ana GV. This model explains
the connection between the GV shell and GvpC and highlights
the structural conservation of GVs between diverse species.
Finally, we extend our study with biochemistry and computa-
tional modeling to corroborate our model and explore its implica-
tions for GV engineering.
RESULTS
Molecular architecture of GVs
Ana GVs are long, cone-tipped cylinders with diameters of 85 ±
4nm
7
and lengths of 519 ± 160 nm
6
(
Figures 1
A and 1B).
Although GVs have apparent helical symmetry, they are prone
to deformation in thin ice (
Figure S1
) and are therefore intractable
for cryoelectron microscopy (cryo-EM) helical processing. For
this reason, we decided to use cryo-ET. However, cryo-ET anal-
ysis of GVs presents its own challenges. We observed that GVs
are highly sensitive to electron dose, losing high-resolution
features quickly before deflating and shrinking (
Video S1
). To
mitigate this effect, we limited the total electron dose to

45 electrons/A
̊
2
per tilt series, which is

2.5 times lower than
typically used for high-resolution subtomogram averaging.
25
,
26
We started by examining large-scale structural features.
While the diameter and length of GVs have been character-
ized,
7
,
27
the conical ends and their connection to the cylindrical
body are less studied. Close inspection of individual caps in our
cryo-tomograms revealed a heterogeneous morphology that
deviated from a simple conical structure (
Figures 1
C and 1D).
We observed two elements in the majority of cones: a pointed
closed tip and a rounded transition region between the cone
and cylinder (
Figure 1
D). The height of the conical caps was
59 ± 6 nm, independent of cylinder diameter (
Figure 1
E). The
rounding of the base was more pronounced in GVs with larger di-
ameters, so we also examined cryo-tomograms of Mega GVs,
whose average diameter is

30 nm smaller than that of Ana
GVs. However, Mega GVs showed similar rounding at the cap
transition (
Figure S2
), suggesting that this is a conserved feature
of the structure independent of width.
60
70
80
90
0
20
40
60
80
100
Diameter (nm)
Height (nm)
z = 71
z = 100
CD
E
100
B
A
Figure 1. Molecular architecture of Ana GVs
(A) Schematic of an Ana GV with dimensions annotated.
(B) Representative slices at the indicated z heights from cryo-ET of an individual GV. Inset: enlargement of the area indicated by the black dashed box
. Scale
bars, 50 nm.
(C) Central tomography slices of two conical GV ends with different morphologies. Scale bars, 50 nm.
(D) Enlarged views of the areas indicated by orange (apex) and blue (cone to cylinder transition) dashed boxes in (C). Scale bars, 10 nm.
(E) Distribution of the diameters and heights of conical GV ends; n = 132. The orange dashed line indicates the average height of the cones (59 ± 6 nm).
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Article
Structure
31
, 518–528, May 4, 2023
519
The GvpA spiral reverses polarity in the middle of the
cylinder
The GV shell consists of a low-pitch helix, running the length of the
GV (
Figures 2
A and 2B). Near the middle of the GV, however, the
angle of the helix abruptly inverts. Previously, Waaland and Bran-
ton
28
noticed that one rib in the middle of the GV cylinder appears
to be thicker than the others and suggested that this could be the
growth point, where new GvpA subunits are added. Indeed, this
abnormal rib was clearly visible in our tomograms (
Figure 2
A).
To obtain a better understanding of the rib architecture in that re-
gion, we applied subtomogram averaging, which revealed that the
angle of corrugation is opposite above and below the central rib
(
Figure 2
B). This polarity inversion occurs within one rib, and the
continuity of the spiral is not broken (
Figures 2
B and 2C). We
were unable to distinguish whether the polarity of GvpA subunits
changed relatively gradually within the space of one helical turn or
abruptly from one monomer to the next. We also could not tell
whether additional proteins are present at the inversion point.
By inspecting hundreds of cryo-electron micrographs of GVs
from different species (Ana, Mega, and
Halobacterium salinarum
),
we found that the polarity inversion point is a conserved feature
(
Figure S3
). Although in general the inversion point was near the
middle of the cylinder, in some cases it was located closer to
one end (
Figure S3
A). If it is the nucleation point, then this sug-
gests that GvpA subunits are not always added symmetrically in
both directions. Additionally, we observed some examples where
a GV exhibited different diameters on either side of the inversion
point (
Figure S3
B). While we saw examples in all three species,
it was most frequent and most pronounced in GVs from
H
.
salina-
rum
(Halo).
Subtomogram averaging of the GV shell
To understand the molecular details of the GV structure, we
applied subtomogram averaging to the Ana GV shell in its
native state and after biochemically removing the reinforcing
protein GvpC to produce ‘‘stripped’’ (AnaS) GVs. Initially,
we tried averaging tubular sections of the GVs. However,
because of flattening and the low number of particles, the res-
olution of this approach was limited (
Figure 3
A). As an alterna-
tive, we decided to average only small sections of the shell
with randomly seeded particle centers similar to an oversam-
pling method.
25
,
29
This strategy produced a higher number of
particles and allowed more rigorous 3D classification to re-
move distorted particles. With this method, we produced sub-
tomogram averages of native Ana (
Figure 3
Band
S4
)and
AnaS (
Figure S5
) GV shells with global resolutions of 7.7 A
̊
and 7.3 A
̊
, respectively (
Table S1
;
Figures S4
and
S5
). Despite
high global resolution, our maps manifested a certain degree
of anisotropy with significantly lower resolution in the y direc-
tion (
Figures S4
D and S5D). The particle poses after subto-
mogram averaging indicate that all particles are oriented
outward and consistent with a helical arrangement (
Figure S6
).
Typically, we observed one significant break in the particle
poses per GV, which corresponds to the inversion point.
However, because of the strong effects of missing wedge
artifacts on tubular structures, such as GVs, they typically
appear as two disconnected arches. As a result, we observed
a fraction of misaligned particles in the direction of the
missing wedge. Furthermore, flattening of the GV cylinder
and small variability in diameters could lead to inaccurate
alignment of some particles, resulting in blurring of the struc-
ture, particularly in the y direction, and limiting resolvability of
the secondary structures. Although the GV corrugated struc-
ture has strong features in the x and z directions, there are no
features in the y direction that could aid subtomogram align-
ment. A visual examination of the maps revealed that, despite
the lower resolution, the map for the native Ana GV shell had
higher quality (
Figures 3
F, 3G, and
S4
C). For this reason, we
z=71
z = 100
z=95
A
B
C
180º
Figure 2. Polarity inversion point
(A) Enlargement of the tomographic slices from
Figure 1
B (indicated by the orange dashed box) at different z heights. The blue dashed outlines indicate sections
where polarity changes. Scale bars, 50 nm.
(B) Subtomogram average of the middle region of the GV where the ribs reverse polarity. Arrows denote the rib where polarity is reversed.
(C) Enlarged view of the subtomogram average in (B), highlighting the inversion of the helical assembly.
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Article
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Structure
31
, 518–528, May 4, 2023