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BREAKTHROUGH REPORT

Cryo-EM Structure of Actin Filaments from Zea mays Pollen Zhanhong Ren1,#, Yan Zhang2,#, Yi Zhang1, Yunqiu He1, Pingzhou Du1, Zhanxin Wang1, Fei Sun2,3,4, * & Haiyun Ren1,*

1Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, Center for Biological Science and Technology, College of Life Sciences, Beijing Normal University, Zhuhai 519087, China. 2National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. 3University of Chinese Academy of Sciences, Beijing, China. 4Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. #These authors contributed equally to this work. *Address correspondence to feisun@ibp.ac.cn or hren@bnu.edu.cn.

Short title: Structure of plant actin filaments

One-sentence summary: Our cryo-EM structural data, together with the single-molecule magnetic tweezers analysis, reveal that the plant actin filament from Zea mays pollen is more structurally stable than the rabbit skeletal muscle actin filament.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Fei Sun (feisun@ibp.ac.cn) and Haiyun Ren (hren@bnu.edu.cn).

ABSTRACT

Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform vital roles in key cellular processes. Although large amounts of data on the biochemical activities, dynamic behaviors, and important cellular functions of plant actin filaments have accumulated, their structural basis is elusive. Here, we report a 3.9 Å structure of the plant actin filament from Zea mays pollen (ZMPA) using cryo-electron microscopy. The structure shows a right-handed, double-stranded (two strands running parallel to each other) and staggered architecture that is stabilized by intra- and interstrand interactions. While the overall structure resembles that of other actin filaments, its DNase I-binding loop (D-loop) bends further outward, adopting an open conformation similar to that of the jasplakinolide- or Beryllium fluoride (BeFx)-stabilized rabbit skeletal muscle actin (RSMA) filament. Single-molecule magnetic tweezers analysis revealed that the ZMPA filament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actin filaments have greater stability than animal actin filaments, which might be important for their roles as tracks for long-distance vesicle and organelle transportation.

Key Words: Cryo-electron microscopy; Structure of plant actin filaments; D-loop conformation; Single-molecule magnetic tweezers; F-actin stability.

Plant Cell Advance Publication. Published on October 18, 2019, doi:10.1105/tpc.18.00973

©2019 American Society of Plant Biologists. All Rights Reserved

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INTRODUCTION

The actin cytoskeleton plays vital roles in many fundamental processes including vesicle and organelle

transportation, endo- and exocytosis, and cell division and growth (Breuer et al., 2017; Takatsuka et al., 2018;

Szymanski and Staiger, 2018; Fu, 2015; Li et al., 2018; Romarowski et al., 2018; Uraji et al., 2018). Actin exists

as two states in vivo: globular actin (G-actin) and filamentous actin (F-actin), which are subject to a dynamic

equilibrium of polymerization and depolymerization. In most instances, F-actin is the functional form of actin

proteins. Thus, studying the structure of F-actin is of particular importance for understanding its functional

mechanism. Recently, the evolution of cryo-electron microscopy (cryo-EM) technology has enabled the

determination of filamentous structures of RSMA in different nucleotide states with resolution ranging from 3.3

to 4.7 Å and the structure of jasplakinolide-stabilized malaria parasite Plasmodium falciparum actin 1

(JASP-PfAct1) at 3.8 Å resolution (Galkin et al., 2015; von der Ecken et al., 2015; Pospich et al., 2017; Merino

et al., 2018). In addition to those of eukaryotic F-actins, high-resolution cryo-EM structures of bacterial and

archaeal actin-like filaments have also been revealed in the last few years, including the 3.6 Å resolution

structure of MamK filaments from magnetotactic bacteria (Lowe et al., 2016), the 3.4 Å resolution structure of

AlfA filaments from Bacillus subtilis (Szewczak-Harris and Lowe, 2018) and the 3.8 Å resolution structure of

Pyrobaculum calidifontis crenactin filaments (Izore et al., 2016).

Despite the high protein sequence identity between plant and animal actins (Kandasamy et al., 2012), their

biochemical activities and cellular functions are different (Jing et al., 2003; Ren et al., 1997; Kandasamy et al.,

2012; Rula et al., 2018). However, the structural basis accounting for these differences remains poorly

understood, largely because none of the plant F-actin structures have been resolved.

Here, we report a 3.9 Å resolution structure of Zea mays pollen actin (ZMPA) filaments determined by

cryo-EM and rupture forces of actin filaments measured by single-molecule magnetic tweezers. Our structural

data show that the ZMPA filament resembles jasplakinolide- or Beryllium fluoride (BeFx)-stabilized

mammalian actin filament, implying that plant actin filaments have enhanced stability. Furthermore, the

recorded rupture events of actin filaments confirm that the ZMPA filament has greater mechanical stability than

RSMA.

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RESULTS AND DISCUSSION

Overall Structure

To determine the structure of plant actin filaments, we obtained highly purified proteins of Zea mays (maize)

pollen actin by taking advantage of the high binding affinity between actin and profilin and the ability of the

actin-profilin complex to bind a poly-L-proline column (Supplemental Figure 1A; Reference 7). Protein mass

spectrometry analysis revealed that the ZMPA samples contained five actin isoforms with approximately 98%

protein sequence identity (Supplemental Figures 1B and 1C). The ZMPA samples were subsequently

polymerized into long and straight filaments in vitro and applied to structural studies by cryo-EM.

ZMPA filaments were highly contrasted to show the double-helical nature of the filaments (Supplemental

Figures 2A and 2B). A cryo-EM dataset was collected, and the structure of the ZMPA filament was

reconstructed using a real-space helical reconstruction approach (Figure 1A; Supplemental Movie 1;

Supplemental Files 1 and 2). ZMPA filaments existed as a two-stranded structure composed of staggered actin

subunits, with a refined helical symmetry of –166.77° rotation and 27.5 Å rise per subunit, resembling those of

RSMA and jasplakinolide-stabilized RSMA (JASP-RSMA) filaments (Figures 1A and 1B; References 1,2,4).

The final 3D reconstruction of ZMPA filaments had an overall resolution of 3.9 Å, using Fourier shell

correlation (FSC) = 0.143 gold-standard criterion (Rosenthal and Henderson, 2003) (Figures 1C and 1D). The

current resolution enabled us to build a pseudo-atomic model of the ZMPA filament with accuracy in the

backbone level. A few charged residues (e.g., K115, E197, D246, E272, and D290) lack a clear side chain

density, which might be due to potential radiation damage or to the intrinsic flexibilities of those residues.

The structure of the ZMPA subunit comprises four subdomains (SDs), SD1–SD4, and the D-loop in SD2 and

hydrophobic plug in SD4 (Figure 1B). The ADP bound in the catalytic pocket, the β sheet in SD3 and the

α-helix in SD4 of the ZMPA subunit are clearly resolved, and side chain densities for the bulkier residues are

visible (Figures 1E–G). When amino acid differences were overlaid onto the structure of the ZMPA subunit, the

sequence divergences among the five ZMPA isoforms were mainly located in SD1 and SD4 but not in the

regions important for nucleotide binding or for intersubunit interactions that involve SD2 and the hydrophobic

groove (Supplemental Figure 2C). In addition, the nucleotide-binding pockets are highly conserved among the

structures of ZMPA, PfAct1 and RSMA filaments (Supplemental Figure 3).

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Intersubunit interactions

Similar to other actin filaments, the ZMPA filament is stabilized by interactions between actin subunits of the

same strand (intrastrand) and the opposing strand (interstrand). Intra- and interstrand interaction sites are

involved in the longitudinal and lateral interfaces, respectively (Supplemental Table 1). The internal interface in

the ZMPA actin filament covers similar areas to those in mammalian actin and JASP-PfAct1 filaments,

according to analysis using the PISA (Proteins, Interfaces, Surfaces and Assemblies) server

(http://www.ebi.ac.uk/pdbe/pisa/) (Krissinel and Henrick, 2007) (Supplemental Figure 4). The longitudinal

interface is formed mainly between SD3 of one actin subunit and SD2 and SD4 of the next actin subunit of the

same strand (Figure 2). Similar to the situations in RSMA and JASP-PfAct1 filaments (von der Ecken et al.,

2015; Pospich et al., 2017), there are two similar lock-and-key hydrophobic interaction sites in ZMPA filaments.

At the first hydrophobic contact site, the major hydrophobic patch in the D-loop of ZMPA filaments interacts

with the hydrophobic groove on the adjacent actin subunit and encloses Y171 in SD3 of the neighboring actin

subunit (Figures 2A–C). The second contact site consists of V289 in SD3 and the hydrophobic pocket in SD4 of

the next subunit on the same strand (Figure 2E). Two potential salt bridges between D290 of SD3 and R64 of

SD2 and between R292 of SD3 and D246 of SD4 form an intrastrand connection between two neighboring

subunits (Figures 2D and 2F). These two electrostatic interaction sites were also reported to be highly conserved

in the structures of RSMA (von der Ecken et al., 2015) and JASP-PfAct1 filaments (PDB ID code: 5OGW).

Previous amino acid mutational analyses and the result of Mical-mediated oxidation modification showed that

the intrastrand interactions are important for actin polymerization and stability (Murakami et al., 2010; Wertman

et al., 1992; Hung et al., 2011; Grintsevich et al., 2017). Thus, the abovementioned potential salt bridges in the

intrastrand interface of F-actin, together with the hydrophobic interactions, may provide structural support for

ZMPA polymerization.

In addition to the intrastrand contacts, the interstrand interactions are also important for actin polymerization

and stability (Pospich et al., 2017; Wertman et al., 1992). As in RSMA filaments, there is also a hydrophobic

contact site between residues 196–203 of SD4 and SD1 of the adjacent subunit in the opposite strand of the

ZMPA filament (Figure 3A). In addition, two potential electrostatic contacts are present in the lateral interfaces

of ZMPA filaments, which were also reported in RSMA and JASP-PfAct1 filaments (von der Ecken et al., 2015;

Pospich et al., 2017). The first contact site is formed by the interstrand interaction of the positively charged

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residue R41 of the D-loop with the negatively charged residue E272 of the hydrophobic plug of the opposing

subunit (Figure 3B). The second interaction site involves two residues (E197 and K115) that form a potential

salt bridge (Figure 3C). Previous studies indicated that mutations of these sites would destabilize the

hydrophobic and electrostatic interstrand interactions of the PfAct1 filament and that PfAct1 mutants could

polymerize into long filaments in vitro only in the presence of JASP, an actin-filament-stabilizing agent

(Pospich et al., 2017). Our observation of the interstrand interactions is therefore consistent with the effective

polymerization of ZMPA monomers into long actin filaments in vitro.

The D-loop/hydrophobic groove interface

It has been well documented that the D-loop is one of the major regions involved in the intrastrand contacts

of F-actins and plays a critical role in actin polymerization and stability (Holmes et al., 1990; Fujii et al., 2010;

Galkin et al., 2015; von der Ecken et al., 2015; Pospich et al., 2017; Grintsevich et al., 2017; Oztug Durer et al.,

2010). Compared to the D-loop (residues 39–53) of RSMA, there are two residue substitutions (glutamine to

threonine 43 and serine to alanine 54) in that of ZMPA (Supplemental Figure 1C). In contrast to that of

jasplakinolide-stabilized PfAct1 (Pospich et al., 2017) and of RSMA filaments in the ADP state (Merino et al.,

2018), the D-loop of ZMPA in the ADP state shifts outward with respect to the filament axis (Figures 4A–4C).

Interestingly, the conformation of the D-loop in ZMPA resembles that in JASP-RSMA and that in RSMA

bound to ADP-BeFx (Beryllium fluoride) (Figures 4C–4E; Figure 5; Supplemental Movie 2). Previous studies

showed that JASP-RSMA and RSMA bound to ADP-BeFx are more structurally stable than that in the ADP

state (Visegrady et al., 2004; Kardos et al., 2007; Isambert et al., 1995), while the prominent structural

difference is that the former structures have an open D-loop and the latter has a closed D-loop (Merino et al.,

2018), implying a possible association between the D-loop conformation and filament stability. Therefore, our

data suggest that the ZMPA filament in the ADP state may be more stable and rigid than RSMA and

JASP-PfAct1 filaments in the same nucleotide state. We noted that Chou and Pollard reported closed

conformations of the actin filament with AMPPNP or ADP-Pi (Chou and Pollard, 2019). This may indicate that

small difference between ATP analogs can greatly change the D-loop conformation. Although the ADP-Pi state

is very stable (Fujiwara et al., 2007), the ratio of the open conformation is smaller in the case of ADP-Pi than

that of ADP-BeFx (Merino et al., 2018). Therefore, besides the open conformation being the main factor of the

stability, other factors would contribute, which merits further studies.

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To test the mechanical stability of actin filaments, we stretched a single F-actin using single-molecule

magnetic tweezers (Figure 6A), which is a powerful tool to assess the mechanical characteristics of single

biomolecules in vitro (Hinterdorfer et al., 2009; Yu et al., 2017; Chen et al., 2018; Strick et al., 1996). When the

stretching force increased, the F-actin was elongated until it was broken (Figure 6B). Since the interaction

between biotin and streptavidin (Tsai et al., 2016) and that between actin antibody and actin are much stronger

than the maximum stretching force (90 pN (pico-Newton)) applied to F-actin (Supplemental Movies 3 and 4),

the breakage of a single tether within the measurement range of our study reflects the disruption of a single

F-actin under tension. The disruption force of the ZMPA filament is centered at approximately 37.8 ± 2.2 pN,

which is significantly stronger than that of the RSMA filament (26.5 ± 1.8 pN, mean ± SE) (Supplemental

Movies 5 and 6; Figure 6C). The result indicates that the ZMPA filament is more stable than the RSMA

filament. The rupture force of a single RSMA filament measured in our study is relatively small compared with

the values previously reported (Kishino and Yanagida, 1988; Tsuda et al., 1996; Liu and Pollack, 2002). It may

result from the fact that the stretched actin filaments in previous studies were stablized with phalloidin but those

in our study were not. A previous study shows that phalloidin binds to the intersubunit interfaces of the actin

filament and couples neighboring actin subunits (Mentes et al., 2018). The stoichiometry of binding is one

phalloidin for one actin subunit and phalloidin can enhance F-actin stability (Visegrady et al., 2004; Mentes et

al., 2018). Besides, our results of stretching experiments of F-actin seeds (Supplemental Movie 3 and 4) show

that the interaction strength between the neighboring actin subunits stabilized by phalloidin is greater than 90

pN, which is used as the maximum stretching force in our study. In addition, stretching systems may also

contribute to the difference of rupture force measured in the previous and our studies. The rupture force

reported by Kishino (Kishino and Yanagida, 1988) is about 1/4 of that by Tsuda (Tsuda et al., 1996) for the

actin filament using the same microneedle method. It was thought that the former did not consider the

randomizing effect of the rotational Brownian motion and had system errors in the calibration of needles (Tsuda

et al., 1996). The actin filament is pulled continuously in the microneedle method, similar to that in optical

tweezers. In optical tweezers, a laser is focused on dielectric beads. The beads experience a three-dimensional

restoring force directed toward the focus (Neuman and Nagy, 2008). During the stretching experiment, optical

tweezers move the trap continually to pull the sample and the system does not achieve equilibrium. For

magnetic tweezers, the tension exerted on the samples is dependent on the position of the magnets. In our

measurements, the magnets move step by step to the sample. At each step, a constant force is exerted on the

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filament and the system achieves equilibrium. Therefore, in contrast to the stepwise pulling used in the

magnetic tweezers method, the continuous pulling in the optical tweezers method makes the system deviate

from equilibrium and the greater rupture force may be measured (Dhakal et al., 2013; You et al., 2014).

The conformation of the D-loop implies that the interaction between the D-loop and SD1 of the adjacent actin

subunit may be strengthened in the ZMPA filament. Indeed, there is a clear density connecting the D-loop and

the C-terminal tail of the neighboring intrastrand subunit of ZMPA, while the corresponding density is absent in

the JASP-PfAct1 and RSMA filaments in the ADP state (Figures 4A-4C). In addition, the D-loop/hydrophobic

groove interface is an important recognition site for the binding of many actin binding proteins to F-actin

(Merino et al., 2018; von der Ecken et al., 2016). The previously reported complex structure of mammalian

actomyosin shows that the helix-loop-helix (HLH) motif of myosin enters the hydrophobic groove on the

adjacent actin subunit and contacts the D-loop (von der Ecken et al., 2016; Mentes et al., 2018; Fujii and

Namba, 2017), illustrating that myosin could directly sense the changes occurring in the D-loop/hydrophobic

groove interface. Our model shows that the D-loop/hydrophobic groove interface of ZMPA in the ADP state is

different from that of RSMA in the same state (Figures 4B and 4C; Supplemental Figures 5A and 5C). This

difference may account for the different interaction modes with myosin and other actin binding proteins that

bind to the D-loop/hydrophobic groove interface. This result is consistent with a recent report that the

measurements of enzymatic and motile activities of Arabidopsis myosins using Arabidopsis actins were distinct

from those measured using animal skeletal muscle α-actin (Rula et al., 2018). These abovementioned

characteristics are important for plant actin filaments, as they need to serve as tracks for long-distance vesicle

and organelle transportation processes that generate extensive tensions, which is different from the

microtubule-centric cargo transportation systems in mammalian cells (Hammer and Sellers, 2011; Brandizzi and

Wasteneys, 2013).

In summary, the present structural and biochemical data suggest that ZMPA filaments are more stable than

RSMA filaments, which provides a possible explanation for their functional differences in cells.

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METHODS

Purification and polymerization of ZMPA

Plant actin was purified from pollen collected from maize (Zea mays) plants during July and August 2016 and

July and August 2017 and then stored at -80℃. The cultivar of maize (Zea mays) plants was Longping No. 206.

Maize pollen actin was prepared as previously described (Ren et al., 1997). Purified pollen actin in buffer G (5

mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.01% NaN3, 0.2 mM ATP, and 0.5 mM DTT) was divided into small

aliquots (approximately 100 μL), flash-frozen in liquid nitrogen and stored at -80℃. The polymerization of

freshly frozen plant actin was tested. The plant actin was dialyzed against buffer G with pH 7.0 overnight to

change from alkaline pH to neutral (Sampath and Pollard, 1991) and was polymerized at a final concentration of

9.5 μM by the addition of 10× KMEI (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH

7.0) (Neidt et al., 2008) at room temperature (25℃) for 4 h. To minimize the effect of nonpolymerizable actin

on the electron microscopy studies and mass spectrometry analysis, the polymerized plant actin filaments were

spun down (100,000 ×g at 4℃) and gently suspended in buffer G (pH 7.0) with 1× KMEI. The protein

concentration was determined with Bradford reagent (Bio-Rad), using BSA as a standard.

Mass spectrometry analysis of ZMPA

Mass spectrometry analysis was performed by the Beijing Huada Protein R&D Center Co., Ltd. (Beijing, P.R.

China). The identities of the plant actin filaments were revealed by liquid chromatography-tandem mass

spectrometry (LC-MS/MS) analysis. The Q Exactive mass spectrometry data (Thermo Fisher Scientific) were

searched against the UniProt maize (Zea mays) database using 15 ppm peptide mass tolerance and 20 mmu

fragment mass tolerance.

Grid preparation and image acquisition for cryo-EM

ZMPA filaments (3 μL) were applied to glow-discharged GIG holey carbon grids (R1.2/1.3, 400 meshes). The

grid was flash-frozen in liquid ethane at approximately 100 K using a semiautomatic plunge device (Thermo

Fisher Scientific Vitrobot Mark Ⅳ) with a blotting time of 5.5 s and blotting force of level 2 at 100% humidity,

25℃. Screening for sample and blotting conditions was performed on a Talos F200C 200-kV electron

microscope equipped with a 4K×4K Ceta camera (Thermo Fisher Scientific). Finally, micrographs of plant actin

filaments for structure determination were collected on a direct electron device (Gatan K2 camera) in a 300-kV

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Titan Krios electron microscope (ThermoFisher Scientific) using the automated acquisition software SerialEM

(Mastronarde, 2005). A total of 3,605 micrographs were recorded at a calibrated pixel size of 1.063 Å with a

total dose of approximately 39 e-/ Å2 and a defocus range from 1.2 to 2.0 μm.

Image processing of the cryo-EM data set

In total, 3,605 micrograph movie stacks were collected during 4 sessions of microscopy data collection hours.

Motion corrections and defocus estimations for all these micrographs were performed using MotionCorr2

(Zheng et al., 2017) and GCTF (Zhang, 2016) respectively. Micrographs with ice contamination, poor Thon

rings, large defocus values and other defects, were excluded before filament boxing. A total of 1,540

micrographs were finally selected. These selected micrographs were multiplied by their theoretical contrast

transfer function (CTF) for initial correction of CTF (Galkin et al., 2015). A total of 8,609 ZMPA filaments

were boxed using e2helixboxer.py in the package of EMAN2 (Tang et al., 2007) with a box width of 168 and

77% box overlap. A total of 40,943 segments were generated with a box size of 384, and 2D classification was

calculated to check the diameter distribution and data quality by RELION 2.0 (Reference 9). For the 2D class

averages without any downsize or binning, the secondary structures could be observed from some of the 2D

class averages (Supplemental Figure 2B). Helical parameters were calculated by indexing the layer lines of the

Fourier transform of the ZMPA filament. An initial helical rise of 27.5 Å and twist of -166.77° were obtained

and used as the initial helical parameters for helical reconstruction. We used the real-space helical

reconstruction algorithm IHRSR (Egelman, 2000, 2007), which was integrated into the Spider (Shaikh et al.,

2008) script to perform 3D reconstruction, and 35,684 particles were finally included to obtain a 3.9 Å map

(Figure 1). The helical parameters converged to 27.5 Å for the helical rise and -166.77° for the helical twist. We

divided CTF2 to correct CTF as we had already multiplied CTF at the beginning. We used SPIDER (Shaikh et

al., 2008) to perform postprocessing (B-sharping). The gold-standard FSC curve was calculated by dividing the

particles into halves at the beginning. The local resolution was estimated by ResMap (Kucukelbir et al., 2014).

Model building and refinement of ZMPA filaments

We used the model of actin from Oryctolagus cuniculus (Merino et al., 2018) (PDB ID code: 5OOC) as a

starting model, and first fitted it into the map by Chimera as a rigid body (Pettersen et al., 2004). Then, residues

were mutated to those in the ZMPA sequence (UniProt ID code: B6TQ08), and these poorly fitted regions were

adjusted manually and refined in Coot (Emsley et al., 2010). Further refinement was conducted with Phenix

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(Adams et al., 2011). The geometry parameters for the final model are presented in Supplemental Table 2.

Single-molecule magnetic tweezers analysis

The basic principle of magnetic tweezers was described in previous reports (Hinterdorfer et al., 2009;

Zlatanova and Leuba, 2003). The experimental setup of our single-molecule magnetic tweezers was constructed

in house, as described in the previous study (Chen et al., 2018). In our study, two ends of a single F-actin were

bound to a streptavidin-coated coverslip and an anti-actin-coated superparamagnetic bead (DynabeadTM M-270

Epoxy; Invitrogen) via the biochemical reactions between biotin and streptavidin and the bonds between actin

and anti-actin antibody, respectively, as shown in Figure 6A. Magnetic tweezers were used to stretch the actin

filament vertically by the application of a horizontal magnetic field generated by a pair of magnets (small

NdFeB magnets with a distance of 0.5 mm) to a superparamagnetic bead to which the actin filament was

attached. The superparamagnetic bead was illuminated by a parallel light placed above the magnets. The

interference of the illuminating light with the light scattered by the superparamagnetic bead produced concentric

diffraction rings in the focal plane of the objective placed below the flow cell. The image of the diffraction

pattern was recorded through a microscope objective (Olympus 60×1.4, oil immersion) with an AVT

Giga-Ethernet charge-coupled device (CCD) camera at 200 Hz. The real-time position (x, y, z) of the bead at

various forces was recorded by comparing the diffraction pattern of the bead with calibration images at various

distances from the focal point of the objective. The motion of the superparamagnetic bead tethered to the

coverslip with a linear bio-molecule can be described as an inverse pendulum with a tension in the z direction.

The fluctuations of the bead in the x-y plane are related to the applied force by F=kBTl/<δx2> according to the

equipartition theorem, where l is the average end-to-end extension of the molecule, kB is the Boltzmann constant,

T is the absolute temperature of the environment and δx is fluctuation of the bead in the x direction (Yan et al.,

2004). The force exerted on the sample increases in a mono-exponential relationship with the magnet’s position

in the z direction. Before the real measurements for actin filaments were made, the force calibration was carried

out. Using a well-studied DNA tether (lambda DNA), the positions of the bead in the x-y plane at each magnet

position was recorded and the tensions were calculated according to F=kBTl/<δx2>. The relationship between

tension and magnet position was obtained by fitting the tension and magnet position with the mono-exponential

relationship. In the measurement of actin filaments, the tension was read out directly based on the fitted

mono-exponential relationship in LabView software according to the magnet’s position. In the stretching

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measurements of actin filament, the measurement was initiated when the magnets were 3 mm away from the

surface of the flow cell and quite low tension (<0.5 pN) was exerted on the sample. Then, the magnets were

lowered to 0.1 mm from the surface of the flow cell finally with a step size of 0.02 mm, staying for 2 s at each

step, and the extension of the sample was traced. The corresponding stretching force on the single tether was

thereby increased from 0 to ~90 pN exponentially. The tension was read out by the LabView software.

Stretching measurement of a single F-actin

To anchor the F-actin, the surface of the coverslip needs to be functionalized. First, the coverslip was cleaned

by sonicating multiple times (1 h in detergent, 1 h in ethanol, 2 h with piranha solution, and 10 min in deionized

water). The coverslip was then incubated with 2 mg/mL methoxy-PEG-silane (MW 5,000; Laysan Bio) and 2

µg/mL Biotin-PEG-silane (MW 3,400; Laysan Bio) in 95% ethanol (pH 2.0) at 70℃ overnight. The reference

beads (2 µm polystyrene beads; QDSphere) were pipetted onto the coverslip and placed on a hot plate at 150℃

for 10 min to melt onto the coverslip. Second, the coverslip was made to be part of the flow cell and then

incubated in passivation buffer (10 mg/mL BSA, 1 mM EDTA, 10 mg/mL Pluronic® F-127 surfactant, 3 mM

NaN3, 10 mM phosphate buffer, pH 7.4) overnight at 37℃ to limit the nonspecific interaction between the actin

sample and the coverslip. Following streptavidin treatment, the coverslip was washed with 1× KMEI buffer (50

mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole, pH 7.0) to remove free streptavidin, and then

incubated with the preformed biotin-labeled F-actin seeds (stabilized with phalloidin) for 5 min to allow seeds

to bind to it via the interactions between biotin and streptavidin. After the removal of free seeds by washing the

coverslip with the instant mixed G-actin solution of 0.15 μM G-actin in buffer G (5 mM Tris-HCl pH 7.0, 0.2

mM CaCl2, 0.01% NaN3, 0.2 mM ATP, and 0.5 mM DTT) and 1× KMEI buffer, the instant mixed G-actin

solution of 0.75 μM G-actin in buffer G and 1× KMEI buffer was flowed into the channel for actin

polymerization and elongation from the barbed ends of immobilized seeds. After actin polymerization for 6 min,

the instant mixed solution of anti-actin-coated superparamagnetic beads, 0.15 μM G-actin in buffer G and 1×

KMEI buffer were gently injected into the flow cell, and incubated for 10 min to allow the beads to bind to the

F-actin. Finally, the free beads were removed by washing the coverslip gently with the instant mixed solution of

0.15 μM G-actin in buffer G and 1× KMEI buffer. To prevent the depolymerization of F-actin, the solution used

to wash the channel should contain a low concentration of G-actin (slightly higher than the critical

concentration of actin polymerization). The anti-rabbit α-actin mouse monoclonal antibody (Santa Cruz; Lot #

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D1514) was coated on the superparamagnetic beads used for the stretching measurement of the single RSMA

filament and anti-plant actin antibody (Sigma-Aldrich; Lot # 492635537) for that of the single ZMPA filament

by referring to the protocol of DynabeadsTM Antibody Coupling Kit (Invitrogen).

The breakage of a single tether resulted in the target superparamagnetic bead moving suddenly out of the

viewing area. To ensure that the single F-actin is tethered between beads and the coverslip, the magnets were

rotated 50 turns before the measurements were taken. Those beads with a single filament rotated freely and

were chosen for measurements, while with more than one filament did not rotate freely and were not

considered. If multiple actin filaments are anchored between the coverslip and the superparamagnetic bead, the

actin filaments will be tangled when we rotate the magnets and the bead cannot rotate accordingly, which helps

us to discriminate the bead with a single actin filament anchored. Finally, the breaking force of a single tether

was measured and defined as the disruption force of a single F-actin in our present study. All measurements

were carried out at 25ºC.

Figure Preparation

Multiple sequence alignments of the actin isoforms were performed using Clustal Omega (Sievers et al., 2011).

Figures were prepared using ResMap (Kucukelbir et al., 2014), Chimera (Pettersen et al., 2004), PyMOL

Molecular Graphics System, Version 2.0 Schrödinger, LLC (http://www.pymol.org/) and Adobe Illustrator CC.

Statistical Analysis

Statistical significance was analyzed by unpaired Student’s t test for two group data. The P value is shown in

Supplemental Table 3. The sample size and the significance level of the P value are described in the figure

legend of Figure 6C.

Accession Numbers

Sequence data from this article can be found in UniProt with accession codes of α-actin (P68135), PfAct1

(P86287), and five Zea mays pollen actin isoforms (B6TQ08, B4FRH8, C0HDZ6, B6SI11 and B4F989).

Cryo-EM maps of ZMPA filaments have been deposited into Electron Microscopy Data Bank (accession code,

EMD-9734) and the corresponding atomic coordinates have been deposited into Protein Data Bank (accession

code 6IUG).

13 / 18

Supplemental Data

Supplemental Figure 1. SDS-PAGE and LC-MS/MS (QE) results of ZMPA.

Supplemental Figure 2. Cryo-EM micrographs and analysis of ZMPA filaments.

Supplemental Figure 3. Comparison of the nucleotide-binding sites from various actin subunits.

Supplemental Figure 4. Longitudinal and lateral contacts of actin filaments.

Supplemental Figure 5. The hydrophobic groove in ZMPA filaments.

Supplemental Table 1. The residues predicted to be involved in intersubunit interactions.

Supplemental Table 2. Data collection and refinement statistics.

Supplemental Table 3. Statistical Analyses Results.

The following Supplemental Data Movies and were submitted to the Data Dryad Repository and are available

at https://doi.org/10.5061/dryad.k0p2ngf42.

Supplemental Movie 1. Cryo-EM reconstruction of ZMPA filament.

Supplemental Movie 2. The ZMPA filament adopts an open-D-loop conformation.

Supplemental Movie 3. The binding strength between the anti-plant actin antibody and ZMPA.

Supplemental Movie 4. The binding strength between the anti-rabbit α-actin antibody and RSMA.

Supplemental Movie 5. The stretching measurement of a single ZMPA filament.

Supplemental Movie 6. The stretching measurement of a single RSMA filament.

Supplemental Movie Legends.

Supplemental File 1. Map of ZMPA filament.

Supplemental File 2. Validation report from wwPDB.

ACKNOWLEDGMENTS

We thank Prof. Edward H. Egelman from University of Virginia for his generous help with the data analysis for

the project. We thank Prof. Yun Xiang, Prof. Li Zhu, Bin Yuan, and Dr. Xia Deng from Lanzhou University for

their support and advice for this research program. Cryo-EM sample preparation and data collection were

carried out at the Center for Biological Imaging (CBI, http://cbi.ibp.ac.cn), Core Facilities for Protein Science,

at the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS). We thank Dr. Xiaojun Huang, Wei

14 / 18

Ding, Boling Zhu, Tongxin Niu, and other staff members at the Center for Biological Imaging (CBI, CAS) for

their support during data collection and image processing. We thank Dr. Wei Li from Institute of Physics in

Chinese Academy of Sciences (CAS) for his advice on the single-molecule magnetic tweezers analysis. This

work was supported by grants from the National Natural Science Foundation of China (91854206 and

31770206 to H.Y.R.; 31770794 to Yan Z.) and from the Ministry of Science and Technology of China

(2013CB126902 to H.Y.R. and 2017YFA0504700 to F.S.).

AUTHOR CONTRIBUTIONS

H.Y.R. and Z.H.R. conceived and coordinated the project. The isolation and purification of ZMPA and structural

analysis was performed in Haiyun Ren’s lab and the work for image processing, model building and refinement

was performed in Fei Sun’s and Edward H. Egelman’s labs. Z.H.R., Y.Q.H., P.Z.D. and H.Y.R. purified the

ZMPA from maize pollen. Z.H.R. performed cryo-EM sample preparation, data collection and preliminary data

processing. Yan Z. performed image processing, model building and refinement. Z.H.R., F.S. and H.Y.R.

analyzed the structure. Z.H.R. performed the single-molecule magnetic tweezers analysis. Z.H.R. prepared the

figures, tables and video of this paper. Z.X.W. helped with the structural analysis and provided suggestions for

the revision of figures. The manuscript was written and revised by Z.H.R., Yan Z., Yi Z., H.Y.R. and F.S.

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Figure 1. Cryo-EM structure of ZMPA filaments.

(A) Stereo view of the three-dimensional (3D) reconstruction. The five central fitted actin subunits are shown (SU-A:

coral; SU-B: green; SU-C: turquoise; SU-D: blue; SU-E: red).

(B) Front and back view of actin subunit A (SU-A) with its density. The four subdomains (SD1–SD4) of SU-A are shown

in gray, tan, salmon and coral, respectively. The D-loop (purple) and hydrophobic plug (yellow) are located at SD2 and

SD4, respectively.

(C) The cryo-EM density used to build the model of ZMPA filaments, colored according to the local resolution of the

map.

(D) FSC curve for final ZMPA reconstruction indicates the two correlations (black and red dashed lines). One

correlation is between the half-datasets (black line), and the other is between the model and the map (red line).

(E) A close-up view of ADP in the nucleotide-binding cleft of actin shown with respective densities.

(F) and (G) A close-up view of the β sheet in subdomain 3 (SD3) and α helix in subdomain 4 (SD4) of SU-A.

Figure 2. The intrastrand interactions in ZMPA filaments.

Surfaces are colored from high (yellow) to low (white) hydrophobicity. Subunit B (SU-B) and Subunit D (SU-D) are

shown in their respective color. Two important longitudinal interfaces will be formed by SU-B and subdomain 2 (SD2)

of SU-D (A–D) and by SU-B and subdomain 4 (SD4) of SU-D (E–F). (E–F) is a 180° right-handed rotation of (A–D)

using the longitudinal axis of rotation.

(A) and (B) SU-B and SU-D are displayed as surface and ribbon representations, respectively. Protein residues are

in stick representation colored by element in gray for carbon and yellow for sulfur atoms. (A) is an zoom-in display of

the interface formed by SU-B and D-loop of SU-D. (B) is a 120° left-handed rotation of (A) using the longitudinal axis

of rotation. Side view (A) and front view (B) show the interaction of the D-loop with the hydrophobic groove of the

neighboring F-actin subunit in ZMPA filaments. The D-loop encloses tyrosine 171 of the adjacent subunit.

(C) and (E) SU-B and SU-D are displayed as ribbon and surface representations, respectively. Protein residues are

in stick representation colored by element in gray for carbon and red for oxygen atoms. (C) is an enlarged display of

the same area as (A). (E) is an zoom-in display of 40° left-handed rotation of the interface formed by SU-D and

subdomain 3 (SD3) of SU-B using the lateral axis of rotation. Side view (C) shows the interaction of the D-loop with

SD3 of the neighboring F-actin subunit in ZMPA filaments. The Y171 in SD3 inserts into the hydrophobic D-loop (C),

and V289 in SD3 inserts into the hydrophobic groove of SD4 of the neighboring subunit (E), resembling two lock-and-

key hydrophobic interaction sites.

(D) and (F) are the enlarged display of the interface formed by SD3 of SU-B and SU-D. Two potential electrostatic

contacts are formed in the intrastrand interface. The backbones of the charged residues involved in the interactions

are shown as red (negative charge) and deep sky blue (positive charge) spheres.

Figure 3. The interstrand contacts in ZMPA filaments.

(A–C) Actin subunit B (SU-B), subunit C (SU-C) and subunit D (SU-D) are shown in ribbons in the color of the

respective subunit.

(A) Surfaces are colored from high (yellow) to low (white) hydrophobicity. (A) is an zoom-in display of 90° left-

handed rotation of the interface formed by SU-C and SU-D using the longitudinal axis of rotation. Protein residues

are in stick representation colored by element in gray for carbon and red for oxygen atoms. The interstrand

hydrophobic contact in ZMPA filaments is mediated by residues 196–203 in SD4 and SD1 of the neighboring

interstrand subunit C (SU-C).

(B) and (C) The potential electrostatic interactions are formed in the interstrand interface. The backbones of the

charged residues involved in the interactions are shown as red (negative charge) and deep sky blue (positive charge)

spheres.

Figure 4. The ZMPA filament shows an open D-loop conformation.

(A–E) Visualization of actin subunit B (SU-B), subunit D (SU-D) and their corresponding densities in longitudinal

interfaces of the jasplakinolide-stabilized P. falciparum actin 1 (JASP-PfAct1; PDB ID code: 5OGW) (A), rabbit

skeletal muscle actin (RSMA; PDB ID code: 5ONV) (B), Zea mays pollen actin (ZMPA) (C), jasplakinolide-stabilized

rabbit skeletal muscle actin (JASP-RSMA; PDB ID code: 5OOC) (D), and rabbit skeletal muscle actin (RSMA)

filaments bound to ADP-BeFx (PDB ID code: 5OOF) (E). The jasplakinolide and its corresponding density are not

shown in (A) and (D). SU-B and SU-D of JASP-PfAct1 are shown in orange and magenta, those of RSMA in cyan

and dark gray, those of ZMPA in green and blue, those of JASP-RSMA in orchid and turquoise and those of RSMA

bound to ADP-BeFx in salmon and brown, respectively. The C-terminal tail (the last amino acid in the C-terminus) of

SU-B is labeled with a black star. The D-loop is bent farther outward with respect to the filament axis in ZMPA (C)

than in JASP-PfAct1 (A) and in RSMA (B).

(C) There is a clear additional density between the D-loop and the C-terminal tail of the neighboring actin subunit in

the ZMPA filament (indicated by the red ellipse). The state of the D-loop in the ZMPA filament is similar to that in the

JASP-RSMA filament (D) and in the RSMA filament bound to ADP-BeFx (E).

Figure 5. Two distinct D-loop states of actin subunits.

When actin subunit Ds (SU-Ds) from five kinds of F-actin are aligned, a magnified view of the boxed D-loops shows

that the ZMPA subunit adopts an open D-loop state similar to that of JASP-RSMA and RSMA bound to ADP-BeFx,

while JASP-PfAct1 and RSMA show a closed D-loop state.

Figure 6. The ZMPA filament resists a greater stretching force than RSMA.

(A) Schematic setup of the magnetic tweezers used in the study (not to scale). The anti-actin-coated magnetic bead

binds to the actin filament that is tethered to a coverslip through interactions between the preformed biotin-labeled

F-actin seed (stabilized with phalloidin) and streptavidin, which forms a single tether. The stretching force imposed

on a magnetic bead will decrease or increase when the permanent magnets are moved up or down.

(B) The stretching curves of rabbit skeletal muscle actin (RSMA) (blue) and Zea mays pollen actin (ZMPA) (red)

filaments. The time-force (upper), time-extension (middle) and force-extension (lower) curves of a single actin

filament are shown. The rupture force of a single ZMPA filament is 35 pN, which is 9.5 pN greater than that of a

single RSMA filament.

(C) Statistical analysis of the disruption force of RSMA and ZMPA filaments. The averaged disruption force of the

RSMA filament is 26.5 ± 1.8 pN (mean ± SE) and that of the ZMPA filament is 37.8 ± 2.2 pN (mean ± SE). ***,

p-value < 0.001, as determined by a two-tailed Student’s t-test. n=75 for RSMA and n=50 for ZMPA.

DOI 10.1105/tpc.18.00973; originally published online October 18, 2019;Plant Cell

Zhanhong Ren, Yan Zhang, Yi Zhang, Yunqiu He, Pingzhou Du, Zhanxin Wang, Fei Sun and Haiyun RenCryo-EM Structure of Actin Filaments from Zea mays Pollen

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