Resource

Single-Unit Imaging of Membrane ProteinEmbedded Nanodiscs from Two Oriented Sides by
High-Speed Atomic Force Microscopy
Graphical Abstract

Authors
Takamitsu Haruyama,
Yasunori Sugano, Noriyuki Kodera, ...,
Yoshiki Tanaka, Hiroki Konno,
Tomoya Tsukazaki

Correspondence
hkonno@se.kanazawa-u.ac.jp (H.K.),
ttsukazaki@mac.com (T.T.)

In Brief
Haruyama et al. established a procedure
for the observation of membrane proteinsin nanodiscs by high-speed AFM in realtime. Using the two-dimensional crystalof streptavidin as well as mica as a solid
support surface, dynamic structuralchanges of membrane proteins in the lipid
environment were visualized from two
distinct angles.

HighlightsdMembrane protein-embedded nanodiscs were used for highspeed (HS)-AFM observationdNanodiscs in an end-up and side-on orientation wereobserved by HS-AFM in real timedHS-AFM combined with nanodiscs is a powerful tool for
membrane protein dynamics

Haruyama et al., 2019, Structure 27, 152–160
January 2, 2019 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.str.2018.09.005

Structure

Resource
Single-Unit Imaging of Membrane Protein-Embedded
Nanodiscs from Two Oriented Sides
by High-Speed Atomic Force Microscopy
Takamitsu Haruyama,1,4 Yasunori Sugano,1,4 Noriyuki Kodera,2 Takayuki Uchihashi,3 Toshio Ando,2 Yoshiki Tanaka,1
Hiroki Konno,2,* and Tomoya Tsukazaki1,5,*
1Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara 630-0192, Japan
2Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan
3Department of Physics, Nagoya University, Nagoya 464-8602, Japan
4These authors contributed equally
5Lead Contact*Correspondence: hkonno@se.kanazawa-u.ac.jp (H.K.), ttsukazaki@mac.com (T.T.)
https://doi.org/10.1016/j.str.2018.09.005

SUMMARY

Membrane proteins play important roles in various
cellular functions. To analyze membrane proteins,
nanodisc technology using membrane scaffold proteins allows single membrane protein units to beembedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic
force microscopy (HS-AFM) have enabled us tomonitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report
HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein
complexes, SecYEG complex and MgtE dimer. The
observed images showed single particles of membrane protein-embedded nanodiscs in an end-up
orientation whereby the membrane was fixed parallelto the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically
fixed to the solid surface, enabling the elucidationof domain fluctuations in membrane proteins. This
technique provides a basic method for the high-resolution imaging of single membrane proteins by
HS-AFM.

INTRODUCTION
Many membrane proteins, including drug targets, show inherent
activity when properly embedded in specific lipid bilayers. Previous atomic force microscopy (AFM) studies of membrane proteins have employed lipid bilayers spread onto the measurement
surface; these lipid bilayers have been prepared from extracted
cell membrane fractions or membrane protein-reconstituted
liposomes (Bahatyrova et al., 2004; Fotiadis et al., 2003; Ruanet al., 2017; Seelert et al., 2000; Shibata et al., 2010). In somecases, purified membrane proteins solubilized by detergent
were directly reconstituted into the supported lipid bilayer onto
152 Structure 27, 152–160, January 2, 2019 ª 2018 Elsevier Ltd.

a supporting solid surface, such as mica and glass (Milhiet
et al., 2006; Sumino et al., 2017). Therefore, the orientation of
AFM observations for membrane protein dynamics is fixed toonly one side (Figure S1A). To overcome this limitation, a monitoring system that can be used to visualize both sides of themembrane is needed for dynamic analyses of certain membraneproteins (Figures S1B and S1C). For example, in many membrane proteins, such as those involved in signal transduction,functional domains on both sides undergo mutual structuralchanges. Moreover, steric hindrance due to the large size of precursor substrate proteins may prevent protein translocation
across the spread membrane. Hence, sufficient space on bothsides is necessary to monitor the movement of precursor proteins and evaluate conformational changes in domains on bothsides simultaneously. Nanodiscs stably hold the disc-like lipid
bilayer with minimal diffusion of the lipids, and provide sufficientspace on both sides of the membrane when placed in a side-onorientation (Rouck et al., 2017; Bayburt et al., 2002). Additionally,
since nanodiscs can stably retain one functional unit of themembrane protein embedded in the membrane, they are suitable for single-molecule dynamic observation in real time byhigh-speed (HS)-AFM (Kodera et al., 2010; Miyagi et al., 2016;
Shibata et al., 2017; Uchihashi et al., 2011). Single-unit imaging
of membrane protein-embedded nanodiscs loaded onto solid
surfaces may be achieved from various orientations using
HS-AFM under appropriate conditions. Here, two membraneproteins, i.e., the protein-conducting channel SecYEG (Tanaka
et al., 2015) and Mg2+ transporter MgtE (Hattori et al., 2007),
were embedded into nanodiscs and observed by HS-AFM
from two oriented sides.
The evolutionarily conserved SecY/SecE/SecG membraneprotein complex (SecYEG) forms a complex with the cytosolic
motor SecA ATPase to drive protein translocation (du Plessis
et al., 2011; Rapoport et al., 2017; Tsirigotaki et al., 2017). The
proton-driven motor SecDF also facilitates protein translation
at the extracytoplasmic side (Tsukazaki, 2018), and the minimum
necessary protein translocation activity can be achieved only
by SecA and SecYEG embedded in the lipid bilayer (Akimaru
et al., 1991). Therefore, we previously determined the crystal
structures of SecYEG and SecA from Thermus thermophilus, aA

SecYEG-ND

B㻍SecYEG-ND

120

13 nm

SecYEG

7.7 ± 0.6 nmCbiotin-tag

0
0

10 nm

SecYAEG-ND

4812Height (nm)SA 2D crystals
SecA

D

㻍
SecYAEG-ND

120

13 nm

16

Counts10.9 ± 0.8 nm

60

0
0

0 nm 10 nm
E

0 nm 10 nm
SecYAEG-ND

13 nm

F

SecYEG
ecYEG
ec
16 S

4
8
12
Height (nm)SecYAEG-ND

13 nm

9.6 nm㻍

0 nm

7.0 nm60

㻍CountsNanodisc (ND)

SecYEG-ND

SecYAEG-ND
S

12 nm

12 nm
1

0 nm

0 nm

Model of SecYAEG-ND

Simulated AFM image

SecYEG

11 nm

SecYAEG-ND

SecA

0 nm 10 nm
G

0 nm

SecYAEG-ND

0 nm

SecYAEG-ND
15 nm

14 nm

10 nm

10 nm

0 nm

SecYAEG-ND
14 nm

10 nm

0 nm

10 nm

(legend on next page)

Structure 27, 152–160, January 2, 2019 153

thermophilic bacterium (Tanaka et al., 2015; Tsukazaki et al.,2008; Vassylyev et al., 2006), and demonstrated that the SecYA/SecE/SecG complex (SecYAEG), which includes the fusion
protein SecY-SecA, retains the ability to mediate protein transport (Sugano et al., 2017). Moreover, the single-unit SecYAEG
complex embedded in a nanodisc (SecYAEG-ND) is strongly
associated with a precursor substrate protein (Sugano et al.,2017; Taufik et al., 2013).
RESULTSSecYEG-NDs and SecYAEG-NDs in the End-Up
Orientation
In this study, we first used HS-AFM to observe the SecYEGreconstituted nanodiscs (SecYEG-NDs) on mica surfaces but
could not distinguish the orientation of the nanodiscs, mainly
owing to the small size of the observed spherical particles,with diameters of 10 nm, as well as nonspecific binding. Hence,
we prepared samples in which all spherical SecYEG-NDs were in
the end-up orientation. Purified SecYEG was labeled with biotin
at two positions on the periplasmic loops using a doublecysteine mutant (Figure S2), embedded in nanodiscs, andanchored onto the two-dimensional crystal of streptavidin(Yamamoto et al., 2009) (Figure 1A). The nanodiscs were presumed to be oriented as shown in Figure 1B (right). HS-AFM
observations showed the presence of 10-nm spherical objects
on the two-dimensional crystal of streptavidin (Figures 1A and 1E[left]). The average height of the spherical molecule (7.7 ± 0.6 nm;
n = 277, 277 molecules) was consistent with the height of
SecYEG estimated from the crystal structure (Tanaka et al.,2015), suggesting that the observed particles represented
SecYEG in the nanodiscs (SecYEG-NDs) (Figure 1B). Next, weobserved SecYAEG-NDs and found that the average height(10.9 ± 0.8 nm; n = 305, 305 molecules) was 3.2 nm greaterthan that of SecYEG-NDs, possibly due to the additional SecA
region. This is consistent with the expected height based on
the crystal structure (Zimmer et al., 2008) (Figures 1C–1E [right]).
Images of SecYAEG-NDs showed round X shapes, similar to thesimulated AFM image from the crystal structure (Figure 1F), suggesting that the X-shaped particles are the domains of SecA.
Close-up observations indicated that each SecA transitioned
among several different conformations, as partially shown instructural analyses (Chatzi et al., 2014) (Figure 1G). Although
the differences in SecA particles may reflect the functional andstructural changes in solution, we could not clearly distinguisheach domain of SecA. Notably, the images showed high resolution, particularly considering that SecA particles did not directlyinteract with the supporting surface but were positionedon the nanodisc apart from the surface. If we introduced atleast three cysteine residues per unit or used shorter biotin-maleimide reagents for fixation to reduce the fluctuation of nanodiscs, we would obtain higher-resolution images. Thus, weestablished a procedure in which biotin-labeled SecYEG-NDs
and SecYAEG-NDs were anchored onto the two-dimensional
streptavidin surface in the end-up orientation. Moreover, after
biotin labeling via introduced cysteines, we can use HS-AFM
as an imaging technique to evaluate membrane-embedded proteins from arbitrary orientations.
SecYAEG-NDs in the Side-On Orientation
To explore the samples from another angle, we observed the
SecYAEG-NDs adsorbed on a mica surface. Unexpectedly,
we found similarly shaped particles in the same orientation.
Each particle consists of one small and one large ellipticallyshaped object, both of which were clearly distinguishable basedon cleavage (Figure 2A). The particle shapes are essentiallyconsistent with the simulated AFM image from the crystal structure, indicating that the SecYAEG-NDs are immobilized with aside-on orientation on the mica surface (Figures 2B and 2C).
Furthermore, to reliably confirm the orientation, mixed samples
of SecYAEG-NDs with streptavidins were observed on the mica
surface (Figure 2D). Compared with the condition without streptavidin, the HS-AFM image showed two additional smaller
particles (size of 5 nm) attached to the side of the large
elliptical molecule, implying that the large and small particles
correspond to the SecYEG-ND and soluble SecA regions,
respectively. Previous AFM studies have shown that membraneprotein-embedded nanodiscs are immobilized with the end-up
orientation on mica (Koch et al., 2016; Zocher et al., 2012). In
our case, the flexibility of SecA and the biotin label may hamper
the close interaction between the mica surface and lipid bilayer.
Our observations demonstrate the presence of SecYAEG-NDs
in the side-on orientation on the mica surface and SecYAEGNDs in the end-up orientation on the streptavidin crystal plane
using biotin-labeled samples. Although it is difficult to further
elucidate the molecular mechanisms of Sec translocation
machinery owing to the resolution of the HS-AFM observation
system, nanodisc systems may allow the observation of protein

Figure 1. SecYEG-NDs and SecYAEG-NDs Fixed on Streptavidin Two-Dimensional Crystals in the End-Up Orientation(A) HS-AFM image of SecYEG-NDs on streptavidin two-dimensional (SA 2D) crystals.
(B) Height distribution of SecYEG-NDs (left). The original maximum heights of SecYEG-NDs on SA 2D crystals were obtained from their cross-sectional profiles.
The actual heights of SecYEG-NDs were calculated by subtracting the background (the median height of the lattice surface not containing holes of SA 2D crystals)
estimated from a cross-sectional analysis. The red line represents the single-Gaussian fitting curve (7.7 ± 0.6 nm; n = 277, 277 molecules). Schematic illustrationof SecYEG-NDs fixed on SA 2D crystals (orange lattice pattern) via biotin tags (right). The SecYEG-ND model was constructed based on the crystal structure of
SecYEG (PDB: 5AWW) and the model of nanodisc including membrane scaffold proteins and lipids (PDB: 2MSC). The estimated height of SecYEG is indicated.
(C) HS-AFM image of SecYAEG-NDs on SA 2D crystal.
(D) Height distribution of SecYAEG-NDs (left). The blue line represents the single-Gaussian fitting curve (10.9 ± 0.8 nm; n = 305, 305 molecules). The actual heights
of SecYAEG-NDs were calculated as in (B). Schematic illustration of SecYAEG-NDs fixed on SA 2D crystals using the crystal structure of the SecA-SecYEG
complex (PDB: 3DIN) and nanodisc model (PDB: 2MSC).
(E) Three-dimensional images of (A) and (C).
(F) Simulated AFM image (right) of the SecYAEG-ND structure constructed by the model in (D) (left).
(G) Close-up view of HS-AFM images of SecYAEG-ND particles on SA 2D crystals, showing the fluctuation of the cytoplasmic domains of SecA.
See also Figures S1 and S2.

154 Structure 27, 152–160, January 2, 2019

Figure 2. SecYAEG-NDs Loaded on the Mica
Surface in the Side-On Orientation

A(A) HS-AFM image of SecYAEG-NDs on the mica
surface (left). Cleavages (indicated by white arrows)
show the interface between SecA and the nanodisc.
The SecYAEG-ND models in (B) are fitted to theobserved particles (right).
(B) Simulated AFM image of the SecYAEG-ND model.
(C) Magnified AFM image of SecYAEG-NDs (top) andcross-sectional profiles shown along the red line
drawn in the upper image (bottom).
(D) HS-AFM image of streptavidin-attached SecYAEGNDs via biotin tags.

B

D

C

transport reactions across the membrane from at least two
directions.
MgtE-NDs in the End-Up Orientation
Next, to address whether the system using nanodiscs is applicable to other membrane proteins, we applied this approachto the Mg2+ transporter MgtE. Dimeric MgtE regulates Mg2+ uptake into the cells in response to Mg2+ concentrations in the cytoplasm (Hattori et al., 2007, 2009; Ishitani et al., 2008). When the
Mg2+ concentration in the cytoplasm is low, the assembly of thecytoplasmic region, consisting of N andCBS domains, weakens, inducing structuralchanges in the transmembrane domain for
Mg2+ transport. In contrast, when sufficient
Mg2+ exists, the cytoplasmic region is tightly
packed, blocking Mg2+ uptake. We purified
dimeric MgtE labeled with biotin on the periplasmic loop using a cysteine mutant (Figure S2) and observed MgtE-reconstituted
nanodiscs (MgtE-NDs) anchored onto thestreptavidin crystal in the presence of Mg2+
(Figures 3A–3C and Video S1). The HSAFM images showed spherical structures,consistent with the simulated AFM image
based on the crystal structure (Hattori
et al., 2007). The highest region (averageheight = 9.6 ± 1.0 nm; n = 2,576, 24 molecules) in the center of the nanodisc isconsidered the dimeric MgtE, consistent
with the estimated height. During the observation, the height of the MgtE-NDs wasnearly constant (Figures 3D and 3E), suggesting that MgtE in nanodiscs in solution
in the presence of Mg2+ was a hard,compact spherical structure, similar to the
crystal structure. In contrast, the HS-AFM
images of MgtE-NDs in the absence of
Mg2+ showed a jagged topography, indicating that the cytosolic domains areconstantly fluctuating (Figures 3F and 3G;
Video S2). In the absence of Mg2+, the height
distribution showed two peaks, which werefitted using two Gaussian curves (Figure 3H).
Although the larger average height (10.4 ±2.0 nm; n = 2,156, 21 molecules) wasapproximately similar to the peak observed in the presence of
Mg2+ (Figure 3E), the distribution became broader comparedwith that in the presence of Mg2+ (Figures 3E and 3H). The
peak at 7.8 ± 0.5 nm corresponds to the height of MgtE-ND
without CBS domains (7.4 nm), presumably due to the fluctuation of the cytoplasmic domains, including CBS domains. Thesephenomena of the cytoplasmic region observed by HS-AFM in
the absence of Mg2+ were consistent with the flexibility of the
N and CBS domains, which are distant from each other in the
crystal structure and easily dissociated without Mg2+ (Hattori
Structure 27, 152–160, January 2, 2019 155

BModel of Mg2+-bound MgtE-ND
Side view
Top view

D

MgtE-ND with Mg2+

N

10

10 nm

9.4 nmm

CBS

E

MgtE-ND with Mg2+
16 nm

0.0 s

5
0
0

0 nm 10 nm

20.4 s58.8 s20
40
Time (s)60

MgtE-ND
with Mg2+㻍200

9.6 ± 1.0 nm

160CountsC

MgtE-ND with Mg2+
15

120
㻍

CBS

N

Simulated AFM image

Height (nm)

A

80
40
0

F

G

MgtE-ND without Mg2+
16 nm

0

10 nm

0.0 s

5
10
15
Height (nm)20

MgtE-ND without Mg2+
15

Height (nm)

0 nm

25.8 s105000 nm 10 nm

H

20
40
Time (s)60

MgtE-ND without Mg2+

42.0 s60.0 sCounts160

10.4 ± 2.0 nm
7.8 ± 0.5 nm

120
80
40
0

0

5
10
15
Height (nm)20

Figure 3. Fluctuation of MgtE-NDs in the End-Up Orientation, Depending on Mg2+
(A) Mg2+-bound MgtE-ND models constructed based on the crystal structure of MgtE (PDB: 2YVX) and the nanodisc model (PDB: 2MSC). The N and CBS
domains are shown in cyan and blue, respectively. Side view showing a schematic illustration of MgtE-NDs fixed on SA 2D crystals via biotin tags. The estimatedheight of MgtE is shown.
(B) Simulated AFM image of the Mg2+-bound MgtE-ND model, as shown in (A) (right).
(C) Successive AFM images of Mg2+-bound MgtE-NDs on SA 2D crystals.
(D) Time course of the height of five independent particles of Mg2+-bound MgtE-NDs.
(E and F) Height distribution of Mg2+-bound MgtE-NDs (E). The red line represents the single-Gaussian fitting curve (9.6 ± 1.0 nm; n = 2,576, 24 molecules). The original
maximum heights of MgtE-NDs on SA 2D crystals were the highest point within the particle images of each frame. The actual heights were calculated by subtracting
the background (the highest point within the SA 2D crystals images for each frame). (F) Successive AFM images of Mg2+-unbound MgtE-NDs on SA 2D crystals.
(G) Time course of the height of five independent particles of Mg2+-unbound MgtE-NDs.
(H) Height distribution of Mg2+-unbound MgtE-NDs. The blue line represents the two Gaussian fitting curves (10.4 ± 2.0 nm and 7.8 ± 0.5 nm; n = 2,156,
21 molecules). The actual heights were calculated as in (E).
See also Figure S2; Videos S1 and S2.

156 Structure 27, 152–160, January 2, 2019

A

B

Model ofMg2+-bound MgtE-ND
N

Nanodisc (ND)

Simulated AFM image
MgtE-ND with Mg2+
12 nm

MgtE-ND with Mg2+
17 nm

N
CBS
ND

biotin-tag

CBS

0 nm

10 nm

0 nm

10 nm

C

MgtE-ND with Mg2+
16 nm

0.0 s

10.8 s

22.0 sND
N
CBS

0 nm 10 nm
D

MgtE-ND without Mg2+
16 nm

0.0 s

18.4 s50.4 sND
N CBS

0 nm 10 nm
E

MgtE-ND without Mg2+ on Ni2+-coated mica surface
16 nm

0.0 s

11.2 s33.2 sND

60.0 s

CBS
N

ND
CBS
N

0 nm 10 nm

Figure 4. MgtE-NDs Placed on the Mica Surface in the Side-On Orientation in the Presence and Absence of Mg2+
(A) Side view of the MgtE-ND model (left). Simulated AFM image of the MgtE-ND model (right).
(B) Magnified Mg2+-bound MgtE-ND particles on the mica surface.
(legend continued on next page)

Structure 27, 152–160, January 2, 2019 157

et al., 2007, 2009; Ishitani et al., 2008). Thus, we could place
MgtE-reconstituted nanodiscs in the end-up orientation, similar
to SecYAEG-reconstituted nanodiscs, and detect real-time fluctuations in the cytoplasmic domain of MgtE in nanodiscs.
MgtE-NDs in the Side-On Orientation
We next observed images of MgtE-NDs adsorbed on mica. All
particles showed the elliptical nanodisc architecture not related
to the Mg2+ concentration, whereas the cytoplasmic domains
appeared different depending on the presence of Mg2+ (Figures
4C and 4D; Videos S3 and S4). We recognized the tightly
assembled N and CBS domains in the presence of Mg2+,
consistent with the simulated AFM image of the crystal structure(Figures 4A–4C). In contrast, in the absence of Mg2+, the cytoplasmic regions were highly flexible and ambiguous (Figure 4D).
These observations are also consistent with the aforementioned
results using the two-dimensional crystal. Additionally, weobserved MgtE-NDs on the Ni2+-coated mica surface, whichis positively charged, strengthening the interaction between
the negatively charged cytoplasmic region and the Ni2+-coatedmica (Figure 4E and Video S5). The HS-AFM images showed
clearer cytoplasmic regions in the absence of Mg2+, and successive images demonstrated the association (e.g., at 0.0 and
33.2 s) and disassembly (e.g., at 11.2 and 60.0 s) of the N andCBS domains in solution. Using HS-AFM, we observed the flexibility of the cytoplasmic domain of MgtE from two controlled
directions in real time.
DISCUSSION
In this study, we employed nanodiscs containing membraneproteins that were biotin labeled via introduced cysteine residues for HS-AFM observation. By optimizing the supporting
surface, membrane protein-embedded nanodiscs were immobilized to the end-up or side-on orientation in a well-controlledmanner. The streptavidin crystal surface can specifically andtightly bind to biotin and prevent other nonspecific interactions, thereby allowing for the imaging of uniform particles inthe end-up orientation. The mica surface enabled us toobserve the membrane protein-embedded nanodiscs in theside-on orientation. The nanodisc lipid surface in the side-onorientation was vertically fixed on the mica surface, although
the hydrophilic head groups of lipids are likely to favor interactions with the hydrophilic mica surface. The biotin label andlarge cytoplasmic region of the membrane protein protruding
at both nanodisc lipid surfaces could prevent interactions
between the lipid bilayer and the mica surface reported in previous studies (Koch et al., 2016; Zocher et al., 2012). This combination of HS-AFM and nanodisc technologies enabled us toobserve real-time, dynamic structural changes in membraneproteins in the lipid environment from two distinct orientations.
This method can be used for the dynamic imaging of othermembrane proteins with a large cytoplasmic region, such as

ABC transporters and receptor tyrosine kinases (Endres
et al., 2014; Thomas and Tampe, 2018). Moreover, drastic
conformational transitions in the soluble domains of membraneproteins could be inferred from static snapshots obtained by
crystallography and electron micrography (Gutmann et al.,2018; Ho et al., 2018; Menting et al., 2013; Mi et al., 2017),
and real-time movies such as HS-AFM observation provide
complementary dynamic information regarding the molecularmechanism underlying membrane protein functions. In addition, this method can be applied to elucidate the dynamics
of inherently disordered soluble regions of membrane proteins.
Thus, this strategy may have applications in the analysis ofimportant biological processes involving membrane protein
dynamics to help understand their molecular mechanism indetail.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d EXPERIMENTAL MODEL AND SUBJECT DETAILSd CONTACT FOR REAGENT AND RESOURCE SHARING
d METHOD DETAILSB Preparationof
SecYEG/SecYAEG-reconstituted
Nanodiscs
B Preparation of MgtE-reconstituted Nanodiscs
B HS-AFM Observation
B Simulation of AFM Images
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information includes two figures and five videos and can befound with this article online at https://doi.org/10.1016/j.str.2018.09.005.
ACKNOWLEDGMENTS
We thank K. Abe and S. Suzuki for secretarial assistance and K. Kobayashi
for technical support. This work was supported by the JSPS/MEXT
KAKENHI (grant nos. JP26119007, JP26119003, JP26291023, JP18H02405,
JP17H05669, JP17K19528, JP16K14713, JP15H01537, JP15K06972,
JP15H04360, JP16H00830, JP16H00758, JP15H03540, and JP25112507),
Mitsubishi Foundation, Noguchi Institute, Naito Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, CREST, JST(grant no. JPMJCR13M1), and PRESTO, JST (grant no. JPMJPR12L3).
T. thermophilus DNA was provided by RIKEN BRC, which is participating in
the National Bio-Resources Project of the MEXT, Japan.
AUTHOR CONTRIBUTIONS
T.H., Y.S., and T.T.: conceptualization, manuscript drafting, and investigation;
T.H., Y.S., N.K., T.U., T.A., Y.T., H.K., and T.T.: methodology; H.K. and T.T.:
supervision.

(C and D) Successive AFM images of MgtE-NDs on the mica surface with (C) and without (D) Mg2+.
(E) Successive AFM images of MgtE-NDs on Ni2+-coated mica surface without Mg2+. Schematic illustration depicting conformational transitions in thecytoplasmic domain, containing N and CBS domains, of MgtE-NDs.
See also Videos S3, S4, and S5.

158 Structure 27, 152–160, January 2, 2019

DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: May 2, 2018
Revised: June 6, 2018
Accepted: September 18, 2018
Published: October 11, 2018
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160 Structure 27, 152–160, January 2, 2019STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

BL21(DE3)

Laboratory stock

N/A

Rosetta2(DE3)

Novagen

Cat# 71397

Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteinsn-dodecyl-b-D-maltopyranoside (DDM)

GlyconCat# D97002

Biotin-PEAC5-maleimide

Dojindo

Cat# 344-06391

MSP1D1

Laboratory stock

N/A

E. coli Total Lipid Extract

Avanti Polar Lipids

Cat# 100500

Bio-Beads SM-2 Adsorbents

BIO-RAD

Cat# 1528920

TEV protease

Laboratory stock

N/A

Plasmid : Vector pTV118N

Takara Shuzo

Cat# 3328

Plasmid : Vector pACYCDuet-1

Novagen

Cat# 71147-3

Plasmid : Vector pET-16b

Daimon et al., 2017

N/A

pTT610 (pTV118N-based vector)

AddgeneCat# 101742

pAK24 (pACYCDuet-1-based vector)

AddgeneCat# 101741

T. thermophilus mgtE

RIKEN BRC

Cat# JCM 10941

Kodec 4.5.7.22 (for cross-sectional analysis)

Kodera et al., 2010

N/A

Origin 2018 (for a graph creation and Gaussian fitting)OriginLab

https://www.originlab.com/

Pymol 2.0 (to draw a model of membraneprotein-embedded in nanodisc)https://www.pymol.org/

N/A

IgorPro 6.36 (for high-speed AFM analysis)

WaveMetrics

https://www.wavemetrics.com/

SecYEG crystal structure

Tanaka et al., 2015

PDB: 5AWW

SecA-SecYEG crystal structure

Zimmer et al., 2008

PDB: 3DIN

Model of nanodisc including membrane scaffold
proteins and lipids

Mazhab-Jafari et al., 2015

PDB: 2MSC

MgtE crystal structure

Hattori et al., 2007

PDB: 2YVX

Ando et al., 2001, 2008; Uchihashi
et al., 2012

N/A

Recombinant DNA

Software and Algorithms

Deposited Data

Other
Laboratory-built high-speed AFM

EXPERIMENTAL MODEL AND SUBJECT DETAILSEach E. coli transformant altered from the E. coli strains BL21 (DE3) (Laboratory stock) and Rosetta 2(DE3) (Novagen, Darmstadt,
Germany) was isolated and stored in LB media containing 20% glycerol at 80 C as a glycerol stock.
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to, and will be fulfilled by the Lead Author, Tomoya Tsukazaki(ttsukazaki@mac.com).

Structure 27, 152–160.e1–e3, January 2, 2019 e1

METHOD DETAILS
Preparation of SecYEG/SecYAEG-reconstituted Nanodiscs
Mutations in secY of pAK24 and pTT610 were introduced by site-directed mutagenesis. The resulting plasmids encoded SecY(L2V,
S148C, R208C, R252G)-His6, SecE, and SecG and SecY(L2V, S148C, R208C, R252G)-(GGSG)4-SecA(C648S)1-939-His10, SecE, and
SecG, respectively. SecYEG was purified, as described previously (Tanaka et al., 2015). SecYAEG was prepared by Ni-NTA column
chromatography, as described previously (Sugano et al., 2017). After decreasing the concentration of NaCl to 50 mM using an
Amicon Ultra filter (Ultra-15, MWCO 100 kDa; Merck Millipore, Darmstadt, Germany), a reductant, b-mercaptoethanol (b-ME),
was added to the protein solution with 15 mM. To remove the reductant, the reduced protein solution was purified using a HiTrap
SP HP column (GE Healthcare, Chicago, IL, USA) (Sugano et al., 2017). Biotin-PEAC5-maleimide (Dojindo, Kumamoto, Japan)
dissolved in dimethyl sulfoxide was added to the purified protein at a molar ratio of Biotin-PEAC5-maleimide:SecYEG = 20:1. The
solution was incubated at room temperature for 1 h and then at 4 C for 12 h. To remove excess Biotin-PEAC5-maleimide, the SecYEG
and SecYAEG solutions were loaded on a Superdex 200 Increase 5/150 GL column and a Superose 6 Increase 10/300 GL column(GE Healthcare), respectively, equilibrated with a buffer (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 5% glycerol, and 0.1% n-dodecylb-D-maltoside [DDM]). The labeled Sec complex was reconstituted into nanodiscs as described previously (Sugano et al., 2017).
Preparation of MgtE-reconstituted Nanodiscs
The mgtE gene from T. thermophilus was cloned into modified pET16b (Daimon et al., 2017) and mutated by site-directed mutagenesis. The resulting plasmid, encoding MG-His10-SSGENLYFQGSH-MgtE(A312C), was introduced into Escherichia coli strain Rosetta2(DE3) (Novagen, Darmstadt, Germany). The transformants were cultivated at 37 C to an A600 of approximately 0.7 in LB medium
supplemented with 50 mg/mL ampicillin and 20 mg/mL chloramphenicol. The expression of MgtE was induced with 0.5 mM isopropylb-D-1-thiogalactopyranoside at 20 C for 16 h. The cells were harvested by centrifugation at 5000 3 g for 10 min, and pellets were
resuspended in a buffer (10 mM Tris-HCl [pH 8.0], 1 mM ethylenediaminetetraacetic acid and 0.1 mM phenylmethylsulfonyl fluoride[PMSF]) and disrupted by sonication with a Q500 Sonicator (QSONICA). After centrifugation at 25,000 3 g for 30 min, the supernatant
was ultracentrifuged (138,000 3 g for 60 min at 4 C), and the membrane fraction was collected. The membrane fraction was
solubilized in a buffer (50 mM HEPES-NaOH [pH 7.0], 300 mM NaCl, 20 mM imidazole, 1.5% DDM, and 0.1 mM PMSF). After ultracentrifugation (138,000 3 g for 30 min at 4 C), the supernatant was mixed with Ni-NTA Agarose (Qiagen, Valencia, CA, USA)equilibrated with buffer A (50 mM HEPES-NaOH [pH 7.0], 300 mM NaCl, and 0.1% DDM) containing 20 mM imidazole for 30 min
at 4 C. The resin was washed with buffer A containing 50 mM imidazole and then eluted with buffer A containing 300 mM imidazole.
The His10 tag was cleaved by His-tagged TEV protease, and the sample was reloaded onto the Ni-NTA column equilibrated
with buffer A containing 20 mM imidazole to remove the TEV protease. The flowthrough fraction was collected and b-ME was
added to a final concentration of 15 mM. The mixture was concentrated using an Amicon Ultra filter (Ultra-4, MWCO 50 kDa; Merck
Millipore) and loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated with buffer A. The labeling of BiotinPEAC5-maleimide was performed as described for SecYEG/SecYAEG preparation. The biotin-labeled protein solution was loaded
on a Superdex 200 Increase 5/150 GL column equilibrated with buffer A.
The protein-lipid mixture containing 6.35 mg/mL biotin-labeled MgtE, 7.8 mg/mL MSP1D1 (a membrane scaffold protein), and
8 mg/mL E. coli phospholipids (Avanti) in buffer A was gently mixed at 4 C for 1 h. To remove the detergent, Bio-Beads SM2(Bio-Rad, Hercules, CA, USA) were added to the mixture. After gently mixing at 4 C overnight, the mixture was filtered using
Centrifugal Filters PVDF 0.22 mm (Millipore) and then loaded on a Superdex 200 Increase 10/300 GL column equilibrated with
50 mM HEPES-NaOH [pH 8.0] and 300 mM NaCl.
HS-AFM Observation
HS-AFM imaging of SecYEG-NDs, SecYAEG-NDs, and MgtE-NDs was performed using a laboratory-built HS-AFM, as previously
described (Ando et al., 2001, 2008; Uchihashi et al., 2012). The HS-AFM was equipped with small cantilevers (BL-AC10DS-A2 or
BL-AC7DS-KU4; Olympus, Tokyo, Japan) and operated in the tapping mode. The free oscillation amplitude was approximately
1.5 nm, and the set-point amplitude was 80 90% of the free amplitude. Imaging rate, scan size, and feedback parameters were
optimized during observation to enable visualization with the minimum tip force.
Prior to observations of the SecYEG-NDs, SecYAEG-NDs, and MgtE-NDs on SA 2D crystals, streptavidin molecules were
two-dimensionally crystallized on the mica surface as previously described (Yamamoto et al., 2009), except SA 2D crystals wereformed within 15 30 min of incubation. SecYEG-NDs and SecYAEG-NDs were incubated for 5 min on SA 2D crystals after
SecYEG-ND and SecYAEG-ND samples were diluted in buffer A (50 mM HEPES-NaOH [pH 7.0] and 300 mM NaCl). The
surface of SA 2D crystals was then washed with buffer (A) HS-AFM imaging was performed in buffer A for SecYEG-NDs or buffer
B (50 mM HEPES-NaOH [pH 7.0], 300 mM NaCl, and 100 mM KCl) for SecYAEG-NDs. To prepare Mg2+-bound MgtE-NDs, purified
MgtE-NDs were diluted in buffer C (50 mM HEPES-NaOH [pH 7.0], 300 mM NaCl, 100 mM KCl, and 50 mM MgCl2), incubated for
5 min on SA 2D crystals, and then washed in buffer (C) HS-AFM observations were carried out in buffer (C) After imaging Mg2+-boundMgtE-NDs, the surface was washed in buffer B, not including MgCl2, to observe Mg2+-unbound MgtE-NDs. HS-AFM imaging was
performed in buffer (B)For observation on mica, SecYAEG-NDs were incubated for 5 min on the mica surface after SecYAEG-ND samples were diluted
into 50 mM HEPES-NaOH [pH 7.0] and 100 mM NaCl. The mica surface was then washed in 50 mM HEPES-NaOH [pH 7.0]. HS-AFM
e2 Structure 27, 152–160.e1–e3, January 2, 2019

imaging was carried out in 50 mM HEPES-NaOH [pH 7.0]. To address the orientation of SecYAEG-NDs on the mica surface, 100 nM
streptavidin in 50 mM HEPES-NaOH [pH 7.0] and 100 mM NaCl was loaded on the mica surface after imaging SecYAEG-NDs. Afterincubation for 5 min, the surface was washed in 50 mM HEPES-NaOH [pH 7.0] and observed in 50 mM HEPES-NaOH [pH 7.0] using
HS-AFM. To prepare Mg2+-bound and Mg2+-unbound MgtE-ND samples on mica, purified MgtE-NDs were diluted in 50 mM HEPESNaOH [pH 7.0] with or without 50 mM MgCl2. Mg2+-bound and Mg2+-unbound MgtE-NDs were incubated for 5 min on the mica surface and then washed in 50 mM HEPES-NaOH [pH 7.0] with or without 50 mM MgCl2. HS-AFM imaging was carried out in 50 mM
HEPES-NaOH [pH 7.0] with or without 50 mM MgCl2. To observe the cytoplasmic domain of MgtE-NDs, Mg2+-unbound MgtE-NDs
were loaded on the mica surface coated with Ni2+ ion as follows. First, 100 mM NiSO4 was deposited on the mica surface andincubated for 5 min. The surface was then successively washed in MilliQ water and 50 mM HEPES-NaOH [pH 7.0]. Mg2+-unboundMgtE-NDs were incubated for 5 min on the Ni-coated mica surface and then washed in 50 mM HEPES-NaOH [pH 7.0]. HS-AFM
observation was performed in 50 mM HEPES-NaOH [pH 7.0].
Simulation of AFM Images
Simulated AFM images of SecYAEG-NDs and MgtE-NDs were calculated using an AFM probe modeled by a cone shape with aradius of 0.5–1 nm and half cone of 10 as previously described (Kozai et al., 2017; Uchihashi et al., 2011). The source code for
Igor Pro 6.36 (Wave Metrics Inc., Lake Oswego, OR, USA) is available on request. The structural models of SecYAEG-ND and
MgtE-ND structures were manually constructed by incorporating the SecA-SecYEG crystal structure (PDB: 3DIN) and MgtE crystal
structure (PDB: 2YVX) into the nanodisc model (PDB: 2MSC) (Mazhab-Jafari et al., 2015) using Pymol (Schrodinger). The calculated
simulation images were smoothed by fast-Fourier transform frequency filtering (cut-off wavelength of 0.77–2.50 nm).
QUANTIFICATION AND STATISTICAL ANALYSISThe single Gaussian curve or two Gaussian curves fitting was performed using Origin2018 (OriginLab, Northampton, MA, USA) to
calculate the average height of molecules. N is the number of counted height data as described in figure legends of Figures 1 and 3.
DATA AND SOFTWARE AVAILABILITY
HS-AFM image acquisition and analysis in this paper was carried out by laboratory-developed software based on Igor Pro 6.36 (Wave
Metrics Inc., Lake Oswego, OR, USA). The source codes are available upon reasonable request. Kodec 4.4.7.39 (the latest version of
Kodec 4.5.7.22 for cross-sectional analysis), a HS-AFM image viewer, coded in Visual C# (Visual Studio 2010, Microsoft, USA) isavailable (Ngo et al., 2015).

Structure 27, 152–160.e1–e3, January 2, 2019 e3

Structure, Volume 27

Supplemental Information

Single-Unit Imaging of Membrane Protein-Embedded
Nanodiscs from Two Oriented Sides
by High-Speed Atomic Force Microscopy
Takamitsu Haruyama, Yasunori Sugano, Noriyuki Kodera, Takayuki Uchihashi, Toshio
Ando, Yoshiki Tanaka, Hiroki Konno, and Tomoya Tsukazakiabcantileverfunctional
domainc
precursor
proteinmembraneproteinmica
functional
domaintranslocatedproteinFigure S1. Schematic interpretation of typical membrane proteins embedded in the lipid bilayer, Related to Figure 1.
a, Conventional measurement of a membrane protein on the mica-supported lipid bilayer by AFM. The membrane protein
directly interacts with the mica surface; observations from only one side of the membrane can be performed. b, A model of amembrane protein possessing functional domains on both sides of the membrane, such as certain kinases. Using the method
described in a, the cooperative conformational changes on both sides of the membrane could not be observed at the sametime. c, Diagram of protein translocation via a protein-conducting channel. To complete the protein translocation across themembrane, sufficient space is required on both sides of the membrane. Using the system in a, steric hindrance of thetranslocated protein hampers the translocation of the substrate protein.

ab

CBS-domain(MolA)

CBS-domain(MolB)

N-domain(MolB)

N-domain(MolA)

SecY

cytoplasm

SecG

SecE

extracytoplasm

S148

R208

A312
(MolA)

A312(MolB)

Figure S2. Positions of Cys mutations in the SecYEG complex and MgtE, Related to Figure 1 and 3.
a, Crystal structure of T. thermophilus SecYEG at 2.7 Å resolution (PDB: 5AWW). b, Crystal structure of dimeric MgtE in thepresence of Mg2+ (PDB: 2YVX). The spheres (violet) show the positions of cysteine substitution for biotin labeling.