Articles

https://doi.org/10.1038/s41594-020-0481-x

Cryo-EM reveals the transition of Arp2/3 complex

from inactive to nucleation-competent state
Mohammed Shaaban   1, Saikat Chowdhury   1 ✉ and Brad J. Nolen   2 ✉

Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by
nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is
unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of
activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucle-
ated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins
in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting
mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex
into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3
complex in diverse cellular processes.

R
egulated actin filament nucleation facilitates the assembly of Arp2/3 complex at the pointed end of the actin filament after nucle-
actin networks with proper timing and localization to drive ation17, making it possible to capture the activated, NPF-bound
cellular processes such as endocytosis and cellular motility1,2. state. Taking advantage of these biochemical properties, we deter-
The Arp2/3 complex is an essential actin filament nucleator that mined by cryo-EM the near-atomic resolution structure of activated
nucleates both linear and branched actin filaments and is conserved S. pombe Arp2/3 complex bound to its nucleated actin filament and
across eukaryotes1,3. The complex exists in an inactive state until the S. pombe WDS protein, Dip1. The structure provides a snapshot
bound by NPFs that stimulate activating structural changes4–11. of the activated conformation of Arp2/3 complex and reveals the
Two subunits in the Arp2/3 complex, Arp2 and Arp3, are termed conformational pathway by which NPFs drive activation.
actin-related proteins (Arps) because of their homology to actin.
Previous experiments suggest that during activation, Arp2 and Arp3 Results
move into, or close to, the side-by-side arrangement of consecutive Structure solution. To create short actin filaments capped with
actin subunits along the short-pitch helical axis of an actin filament, Dip1-bound Arp2/3 complex for single-particle reconstruction, we
hereafter called the short-pitch conformation7–12. In this position, mixed Dip1 with Arp2/3 complex and monomeric actin to nucleate
Arp2 and Arp3 are thought to mimic a filamentous actin dimer, new filaments and added capping protein to block barbed-end elon-
thereby creating a template for new filament assembly. In the inactive gation. Optimized reactions produced many short linear filaments
complex, however, Arp2 and Arp3 do not mimic a filamentous actin with knobs of density corresponding to Arp2/3 complex at one end
dimer13,14. Instead, two core subunits of the Arp2/3 complex, ARPC2 (Extended Data Fig. 1a,b). From these samples we obtained a ~3.9 Å
and ARPC4, form a clamp that holds the Arps in an end-to-end resolution reconstruction of Dip1 bound to activated Arp2/3 com-
(splayed) arrangement distinct from the short-pitch conformation. plex, with the complex anchored to the pointed end of the filament
Furthermore, in the inactive complex, Arp2 and Arp3 adopt confor- that it nucleated (Fig. 1a,b, Extended Data Figs. 1b–d, 2a and 3a,c–l
mations that resemble unpolymerized actin monomers rather than and Table 1). The structure contains the first four actin subunits
filamentous actin subunits15,16. The structural differences between (n, n + 1, n + 2 and n + 3) in the nucleated actin filament. The first
filamentous actin dimers and actin-related proteins in the inactive two actins (n and n + 1) are attached to the barbed end of the Arps
Arp2/3 complex suggest that both intra- and intersubunit changes (Figs. 1a and 2a).
within the complex are required to create a nucleation-competent To understand the structural changes that occur during activa-
state. However, the lack of a high-resolution structure of activated tion, we also determined a ~4.2 Å resolution structure (Fig. 1c) of
Arp2/3 complex has prevented an understanding of how NPFs trig- the inactive S. pombe Arp2/3 complex using a tilted cryo-EM data
ger these changes. collection methodology18 (Extended Data Figs. 1e–h, 2b and 3b and
High-resolution structures of activated Arp2/3 complex have Table 1). This structure is nearly identical to those of the inactive
been unobtainable because the activated complex is recalcitrant Arp2/3 complex from Bos taurus13,19 (Extended Data Fig. 4).
to crystallization and tends to adopt limited orientations in vitri-
fied ice during cryo-EM studies. Recently it was discovered that Arp2 and Arp3 adopt the short-pitch conformation upon activa-
the WISH/DIP/SPIN90 (WDS) family of NPFs activate Arp2/3 tion. Previous low-resolution EM reconstructions and biochemical/
complex to nucleate linear actin filaments3, which are more ame- biophysical assays suggested that Arp2 and Arp3 move into or near
nable to single-particle cryo-EM studies than the branched actin the short-pitch conformation during activation8–12, but the struc-
filaments created during activation by other NPFs. Furthermore, tural rearrangements that allow these subunits to trigger nucleation
unlike WASP family NPFs, WDS proteins remain associated with have remained unclear.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA. 2Institute of Molecular Biology and Department of Chemistry
1

and Biochemistry, University of Oregon, Eugene, OR, USA. ✉e-mail: [email protected]; [email protected]

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Articles Nature Structural & Molecular Biology

a b ACTIVE c INACTIVE
PE PE
ARPC2 Dip1 ARPC2 Dip1 ARPC2 Arp3
Arp3 ARPC3
ARPC2 ARPC3
ARPC4
ARPC5 ARPC4
ARPC5
ARPC4
Arp3 Arp3
Arp2 Arp2
Arp2
ARPC1 Dip1
n
Arp2 n ARPC5
Arp2 ARPC5 ARPC1 ARPC1
ARPC4
n+1 ARPC2 Arp3 ARPC3
Arp3
ARPC3
n+1 ARPC2

n+2
n+2
n+3 n+3 Dip1 Arp2 ARPC4
Arp2

BE ARPC5 ARPC1 ARPC5

BE ARPC1
ARPC4

Fig. 1 | Overview of structures of inactive Arp2/3 complex and activated Arp2/3 complex bound to the actin filament pointed end and Dip1.
a, Reconstructed map (left) and atomic model (right) of actin filament pointed end with bound Arp2/3 complex and Dip1. PE, pointed end; BE, barbed
end. b, Map (top) and atomic model (bottom) of activated Arp2/3 complex structure viewed from the pointed end looking down the helical axis of the
nucleated actin filament. c, Map (top) and molecular model (bottom) of inactive Arp2/3 complex.

In the active structure we present here, we find that the Arps At the Arp–actin long-pitch interfaces, three structural features
form a short-pitch heterodimer that closely mimics the filamen- from the pointed end of each actin subunit insert into three cor-
tous short-pitch actin dimer (Fig. 2a,b, Extended Data Fig. 5 and responding creases in the barbed ends of the Arps, making interac-
Supplementary Video 1). Arp2 is rotated ~9° about an axis orthogo- tions analogous to long-pitch interactions between actin subunits in
nal to the filament axis (Fig. 2b), but remains in close contact with a filament15,16 (Fig. 3). Conformational changes at the barbed ends
Arp3 (~450 Å2 buried). This contrasts with the low-resolution EM of the Arps that occur concurrently with flattening are important
reconstruction of the branch junction, in which Arp3 and Arp2 are in facilitating a subset of these interactions. Specifically, flattening
separated by a wide gap12. The 9° rotation of Arp2 does not cause promotes insertion of the D loop of actins n and n + 1 into the crease
long-range distortions in the nucleated filament, because the actin between subdomains 1 and 3 in the Arps, called the barbed-end
subunits in the Dip1–Arp2/3–filament structure show helical groove (BEG) (Extended Data Fig. 7a,b). Flattening of the Arps is
parameters similar to those reported in high-resolution cryo-EM accompanied by uncurling of the W loop in subdomain 3, which
structures of actin filaments (Fig. 2a)15,16. Besides creating a opens a hydrophobic pocket in the BEG that accommodates Met44
filament-like Arp2–Arp3 heterodimer, movement of Arp2 into the of the actin D loop (Extended Data Fig. 7a–d and Supplementary
short-pitch conformation releases Arp2 from its splayed (inactive) Fig. 1). Flattening also rotates a trio of residues that line the subdo-
position at the barbed end of Arp3 (Fig. 2c). This rearrangement main 1 side of the BEG into position to make a hydrophobic pocket
frees the barbed end of Arp3 to interact with the first actin subunit that binds Val45 in the actin D loop (Extended Data Fig. 8a,b and
(n) in the nucleated actin filament, and is one of multiple structural Supplementary Fig. 1). Finally, flattening pulls the C terminus of
changes that trigger activation. both Arps away from the front of the BEG (Extended Data Fig. 8a–c).
The C terminus of Arp3 is five residues longer than in actin and
Flattening of the Arps gates interactions with the first actin sub- includes hydrophobic residues that pin it to the BEG in some inac-
units. While intersubunit structural changes in Arp2/3 complex jux- tive structures7. In this position, the Arp3 C terminus would block
tapose the Arps like two consecutive subunits along the short-pitch the D loop of actin monomer n from interacting with the Arp3
helical axis of a filament, intrasubunit changes within the Arps are barbed end during activation (Extended Data Fig. 8b). By pulling
also important to create the nucleation-competent state. During the C-terminal extension out of the groove, flattening may allow
activation, both Arps undergo a scissor-like rotation of their small the D loop from monomer n to bind, thereby permitting nucleation.
domain (subdomains 1 and 2) relative to their large domain (subdo-
mains 3 and 4) that moves them from a twisted toward a flattened Nucleotide-binding state of the Arps in activated Arp2/3 complex.
conformation (Fig. 2d,e). This conformational change also occurs In the active structure, both the Arp3 and Arp2 nucleotide-binding
in actin following incorporation into filaments, and is important clefts show density consistent with ATP (Fig. 4). In Arp3 the nucleo-
for facilitating both long- and short-pitch interactions between tide density is weaker than in Arp2, but density that could be attrib-
filamentous actin subunits15,16,20. In the activated structure, Arp2 uted to the γ-phosphate is present in both Arps. In contrast, all four
flattens to the same extent as polymerized actin subunits (Fig. 2d actin subunits in the reconstruction appear to have hydrolyzed
and Extended Data Fig. 6a). In contrast, Arp3 flattens only partially, their bound ATP and released the γ-phosphate (Fig. 4). The appar-
adopting a conformation between the structures of monomeric ver- ent density for the γ-phosphate of ATP in the Arp nucleotide clefts
sus filamentous actin subunits (Fig. 2e and Extended Data Fig. 6a). is consistent with previous biochemical experiments showing that
However, partial flattening allows Arp3 to bury ~200 Å2 more sur- ATP hydrolysis is not required for activation21,22.
face area and make increased interactions with Arp2 that mimic the The presumed stability of ATP in the Arp3 cleft could be
actin–actin short-pitch interface (Extended Data Fig. 6b–d). caused by the intermediate conformational state of Arp3. Distance

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Nature Structural & Molecular Biology Articles
as much as filamentous actin subunits and its equivalent glutamine,
Table 1 | Cryo-EM data collection, refinement and validation
Gln137Arp2, rotates into its proposed activated position within the
statistics
nucleotide-binding cleft (Fig. 4d,e). However, in Arp2 the ATP is
Dip1–Arp2/3–actin Inactive Arp2/3 pushed deeper in the nucleotide-binding pocket, into a position
(EMD-21502, PDB complex (EMD- that probably prevents the γ-phosphate from aligning properly with
6W17) 21503, PDB 6W18) Gln137Arp2, His161Arp2 and the catalytic water molecule for hydroly-
Data collection and processing sis (Extended Data Fig. 9c).
Previous data showed that WASP-mediated branching nucle-
Magnification ×92,000 ×120,000
ation stimulates ATP hydrolysis on Arp2, and probably also on
Voltage (kV) 200 200 Arp3, to trigger branch destabilization and actin network disassem-
Electron exposure (e /Å )
– 2
36.35 44.34 bly21,22,24,25. Given that Arp2 hydrolyzes ATP seconds after branch-
Defocus range (μm) –1.00 to –1.75 –0.70 to –1.30 ing nucleation24, we expected ATP to be hydrolyzed on Arp2, and
possibly also Arp3, in our structure of Dip1-activated Arp2/3 com-
Pixel size (Å) 1.12 0.8757
plex, because we aged filaments for tens of minutes to hours before
Symmetry imposed C1 C1 plunge-freezing to image them. The apparent presence of ATP in
Initial particle images (no.) 3,500,000 3,336,981 Arp2 and Arp3 of the Dip1-activated Arp2/3 complex suggests
Final particle images (no.) 110,433 112,170
that Dip1 may not trigger rapid ATP hydrolysis following activa-
tion. Given the role of ATP hydrolysis in destabilizing branches, this
Map resolution (Å) 3.9 4.2 biochemical feature could contribute to the stability of Dip1 bound
 FSC threshold 0.143 0.143 to activated Arp2/3 complex on the pointed end of nucleated actin
Map resolution range (Å) 3.5–5.5 3.9–6.0 filaments17.
Refinement
Twisting of the clamp rotates a block of subunits into the
Initial models used PDB 3DWL, PDB PDB 3DWL short-pitch conformation. The structures we present here not only
3J8I reveal the key features of the nucleation-competent state, but they
Model resolution (Å) 3.92 4.45 also show how Arp2/3 complex switches to this state. To adopt the
 FSC threshold 0.5 0.5 short-pitch conformation, the complex undergoes a dramatic con-
formational rearrangement in which the center of mass of Arp2
Model resolution range (Å) 2.3–4.0 3.7–4.5
moves 20.5 Å relative to its position in inactive structures (Fig. 2c).
Map sharpening B factor –45.12 –116.22 A previously proposed ‘clamp-release model’ hypothesized that to
(Å2) reach this state, Arp2 releases from the clamp subunits (ARPC2
Model composition and ARPC4)—which remain rigid—and rebinds in a new position
 Nonhydrogen atoms 27,399 12,727 that approximates the short-pitch conformation12. This model pre-
dicts that Arp2 will make a distinct set of contacts with the clamp
 Protein residues 3,483 1,854
in the short-pitch state compared to the splayed state. By contrast,
 Ligands 6 Mg2+, 2 ATP, 4 Mg2+, 2 ATP a second model, based on computational studies, suggested that
ADP, 5 phalloidin Arp2 maintains its contacts with the clamp during activation but
B factors (Å2) that the clamp twists to move Arp2 into the short-pitch confor-
 Protein 102.53 51.05 mation26. The Dip1–Arp2/3–filament structure is incompatible
with the clamp-release model, since Arp2 makes identical contacts
 Ligand 92.30 82.27
to the clamp in the inactive and active conformations (Extended
R.m.s. deviations Data Fig. 10a). Furthermore, we find that the clamp itself is not
 Bond lengths (Å) 0.004 0.004 rigid during the activation process, a prerequisite of the clamp-
 Bond angles (°) 0.967 0.983 release model. Instead, during activation the clamp twists, rotating
its bottom half along with a large block of subunits that includes
Validation
Arp2, the globular portion of ARPC4, ARPC1 and ARPC5, to
 MolProbity score 1.65 1.42 bring Arp2 into the short-pitch position (Fig. 5a,b, Extended Data
 Clashscore 5.63 4.67 Fig. 10b and Supplementary Video 2). The twist axis runs parallel
 EMRinger score 1.39 1.29 to the long helices in the clamp and rotates the block of subunits by
~19° (Fig. 5b).
 Poor rotamers (%) 0.00 0.00
To trigger clamp twisting, Dip1 binds the center of the clamp
Ramachandran plot along a long helix that extends from the globular portion of the
 Favored (%) 95.00 96.96 ARPC4 subunit (Fig. 5c). Its binding mode is similar to the human
 Allowed (%) 5.00 3.04
WDS protein, SPIN90, bound to the inactive Arp2/3 complex
observed in a recent crystal structure27. In the active structure we
 Disallowed (%) 0.00 0.00 present here, Dip1 remains braced against the globular domain of
CaBLAM outliers (%) 4.2 3.8 ARPC4 but changes its register with the extended helix in ARPC4
to pack more tightly against it (Fig. 5c–e). This allows Dip1 to serve
as a wedge that bends the helix and pushes it away from the globular
domain of ARPC4 (Fig. 5c–e and Extended Data Figs. 3l and 10b).
measurements in the active site suggest that partial flatten- Bending of the extended ARPC4 helix triggers the ~19°-clamp twist
ing of Arp3 fails to fully rotate Gln160Arp3 into position near the that rotates Arp2 into the short-pitch conformation. Bending of this
γ-phosphate of ATP, where it is thought to position the hydrolytic helix also appears to indirectly stimulate the intrasubunit domain
water molecule23 (Fig. 4c,d). It is important to note that the limited rotation that partially flattens Arp3 because—once moved into the
resolution of the map increases the uncertainty of these measure- short-pitch conformation—flattening of Arp3 increases its interac-
ments (Extended Data Fig. 9a,b). Arp2, on the other hand, flattens tions with Arp2 (Extended Data Fig. 6).

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a b c
Arp3 PE PE
PE
4 Arp3
2
Arp2
Actin PE
27.2 Å Arp2
–165.5°

27.9 Å Arp2
–168.7° short- Arp3
n
pitch
27.8 Å n+1 1
–166.7° 2

n+2 28.0 Å Actin

–166.8° (n)
4
20.5 Å
28.0 Å n+3
–167.3°

3 BE Arp2
1 splayed
BE
BE
BE

d e
Inactive Inactive
2
Active 4 Active
4
2 2
2

~90° ~90°

1
1

1 3 3
1

Fig. 2 | Arp2 and Arp3 flatten and adopt the short-pitch helical conformation upon activation. a, Ribbon diagram showing the two actin-related subunits
at the pointed end (PE) of the nucleated actin filament. The rise and twist of each subunit is indicated. Black and red arrows denote short- and long-pitch
helical interactions within the filament, respectively. BE denotes barbed end. b, Superposition of Arp2 and Arp3 from the active structure with a short-pitch
actin dimer from an actin filament (PDB 6DJO)16. The axis of rotation (yellow arrow) of Arp2 (9°) as compared to its equivalent actin subunit in the dimer
is shown. Subdomain numbers are shown in gray ovals throughout. c, Movement of Arp2 from the splayed (inactive) to short-pitch conformation.
Blue X’s show potential steric clash between Arp2 in the splayed conformation and the first actin subunit in the nucleated filament (transparent surface).
d, Superposition of Arp2 in the active structure with subdomains 3 and 4 from the inactive structure. Cyan arrows denote flattening motion.
e, Superposition of Arp3 in the active structure with subdomains 3 and 4 from the inactive structure.

a b c
2
4 2
Arp3 4 2
4
Arp2 Actin
(n)

3 3
1 3
αK/β15
αI W C′ αK/β15
W W
αI 1 αK/β15 C′ 1
αI
C′
D loop 61–65 13 61–65
13 2/β
β12/β13 2/β β1 D loop
β1 D loop
2
2 2
4
4 4 Actin
Actin (n + 2)
Actin
(n) (n + 1)

1 1 1
3 3
3

Fig. 3 | Arp2 and Arp3 make filament-like long-pitch interactions with actin. a, Long-pitch interactions between Arp3 and actin (n). The αK/β15 insert is
red. Structural features on the pointed end of actin that interact with the barbed end of Arp3 are blue. b,c, Long-pitch interactions between Arp2 and actin
(n + 1) (b) and between actin (n) and actin (n + 2) (c), with highlighted features color coded as in a. Subdomain numbers are shown in gray ovals. These
interactions are described in detail in Extended Data Figs. 7 and 8.

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Nature Structural & Molecular Biology Articles
a b

ATP?

ATP
Arp3

Arp2 Arp3
ADP
Arp2
Actin
(n)
ADP

Actin
(n + 1) Actin (n) Actin (n + 1)
ADP
Actin
(n + 2)
ADP
Actin
(n + 3)

Actin (n + 2) Actin (n + 3)

c d e
P1
loop 0 P1
Arp3, inactive loop
Arp3, active
T14 em P2
P2 Arp2, inactive loop
–10
Twist angle φ (°)

Se loop
ns em Arp2, active
or T12
lo Actin mon.
op
x4 Actin fil.
D185 –20
x4 D154

Q160 Q137
Sensor loo H161
p
H192
–30
2 4 6 8 10
Distance x4 (Å)

Fig. 4 | Nucleotide-binding states of the Arps and actin. a, Structure of activated Arp2/3 complex with nucleated actin filament in which the bound
nucleotides modeled into the Arps and actin subunits are shown as spheres with yellow carbon atoms. b, Individual maps of bound nucleotides (shown in
gray) with the corresponding nucleotide structures inside the map. c, Close-up view of boxed region in a showing active Arp3 (orange) superposed with
an AMP-PNP-bound actin subunit from actin filament PDB 6DJM (ref. 16; semitransparent cyan) using subdomains 3 and 4. The distance x4 between the
proposed catalytic glutamine (Q160) and the γ-phosphate is shown as a dotted line. d, Plot of distance x4 versus the flattening/twisting angle for inactive
or active Arp2 or Arp3 or monomeric (mon.) or filamentous (fil.) structures of actin. All structures had bound ATP or AMP-PNP. Data points labeled ‘em’
are from the active structure presented here (see Methods for additional details). e, Close-up view of white boxed region in a showing active Arp2 (red)
superposed onto inactive Arp2 from the structure PDB 4JD2 (ref. 19; cyan) using subdomains 3 and 4. Flattening rotates Q137Arp2 into or near its presumed
catalytically activated position.

Discussion is stabilized by intersubunit interactions between the Arps and by

While WASP family NPFs bind to different sites on Arp2/3 com- their interactions with the clamp7 (Extended Data Fig. 10a). In the
plex than Dip1 (refs. 6,11,27–30), biochemical and low-resolution struc- inactive conformation, the barbed ends of the Arps are not config-
tural data suggest that, like Dip1, WASP stimulates the movement ured to make strong long-pitch interactions with actin monomers
of Arp2 into the short-pitch conformation3,8–11. Based on the flex- and Arp2 sterically blocks the barbed end of Arp3, thus preventing
ibility of the clamp we observed here and the requirement to move nucleation. The clamp-twisting conformational change induced by
the Arps into the short-pitch conformation during activation31, we Dip1 binding during activation disrupts the inactive arrangement
expect that clamp twisting is a general feature of activation triggered of the Arps and moves Arp2 into the short-pitch conformation,
by WASP and other NPFs. Given that diverse NPFs regulate Arp2/3 thereby permitting association of actin monomers with the nascent
complex across a broad range of biological functions32, we believe filament.
this conformational change to be a key step in controlling actin Intrasubunit changes are also important for activation, and the
assembly in many different cellular processes. activated structure shows that flattening causes structural rear-
Besides clamp twisting and adoption of the short-pitch confor- rangements at the barbed ends of both Arps that allow them to
mation, the structure of activated Arp2/3 complex reveals the other make increased long-pitch interactions with actin (Fig. 6 (states 3
conformational changes required for activation and, along with pre- and 4) and Extended Data Figs. 7 and 8). Some of these changes—
viously published data, allows us to propose a structural pathway for including W-loop uncurling and realignment of the two sides of
actin filament nucleation (Fig. 6). In the inactive complex, the Arp2 the barbed-end groove—occur in both activated Arps and polym-
and Arp3 subunits adopt a splayed arrangement. This conformation erized actin subunits15,16, thereby constituting an evolutionarily

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Articles Nature Structural & Molecular Biology

a b Inactive Active
ARPC2 Arp3 ARPC3

ARPC2

Dip1

Arp3 Arp3

ARPC4 Arp2

ARPC5 Arp3 ARPC4

Arp2

Inactive Arp2
–56.4° –37.1°
Active

ARPC1

c d e
138.8˚ 132.8˚ R205 Y275
ARPC2
(SPIN90) Dip1 αD Inactive Active
H6-2
239 155 155
239 R158
αD
H5-2 6.2 Å
D278
147 9.4 Å L151
247 147 247 ARPC4
H6-2 H5-2 αD
K150
V281
8.0 Å E148
D284
6.6 Å
N′ ARPC4
ARPC4 R247
D145
D143
R288
Inactive Active

Fig. 5 | Dip1 bends the ARPC4 long helix to twist the clamp and rotate a block of subunits into the short-pitch conformation. a, Comparison of active
and inactive Arp2/3 complex structures. Subunits Arp2, ARPC1, ARPC4 and ARPC5 of the inactive structure are shown in semitransparent surface
representation. Cyan curved arrow denotes direction of rotation during activation of a block consisting of Arp2, ARPC1, ARPC4 and ARPC5. b, The clamp
(subunits ARPC2 and ARPC4, transparent surface) rotates to move the Arps into the short-pitch conformation. c, Comparison of contacts between
SPIN90/Dip1 in inactive (PDB 6DEC) versus active Arp2/3 complex. Cyan arrows show direction of motion of the ARPC4 αD helix following activation.
Red lines show bend angle of ARPC4. d, End-on view of helices H5-2 and H6-2 from Spin90/Dip1 and helix αD from ARPC4 in the active and inactive (PDB
6DEC) structures. e, Interactions between Dip1 (pink), ARPC4 (blue) and ARPC2 (cyan) in the active structure.

conserved conformational switch that controls assembly. As in blocks the D loops of the first two actin subunits (n and n + 1) from
Arp3, residues in the C-terminal tail of both actin and Arp2 are inserting into the barbed-end grooves of the Arps (Supplementary
pulled out of the barbed-end groove and toward the filament heli- Fig. 2). These observations suggest that bound WASP blocks proper
cal axis during flattening (Extended Data Fig. 8). However, it is association of its recruited actin subunits with the barbed ends of
currently unclear whether, like Arp3, movement of the relatively the Arps. Therefore, while bound WASP, like Dip1, stimulates the
short C-terminal tails in Arp2 or actin during flattening gates their short-pitch conformation3,8–10, it probably blocks subsequent acti-
long-pitch interactions with actin subunits. Disorder in inactive vating steps. This hypothesis is supported by recent observations
structures makes it unclear whether the C terminus is sufficiently showing that WASP must be released before nucleation37, and also
long and positioned correctly to block interactions of the D loop of by the observation that when Arp2/3 complex is crosslinked into the
an incoming actin monomer with the barbed-end groove in these short-pitch conformation it is inhibited by WASP-CA8. We speculate
proteins16,19. that by stimulating the short-pitch conformational change without
How Dip1 and other WDS proteins activate Arp2/3 complex blocking filament-like interactions with the first actin monomers,
without a pre-existing actin filament while WASP proteins can- WDS proteins allow activation to proceed without Arp2/3 com-
not remains a key unresolved question, because this biochemical plex binding to actin filaments. During WASP-mediated branching
property allows WDS proteins to initiate the assembly of branched nucleation, actin filaments may stimulate WASP release from the
actin networks3,33,34. We postulate that a key difference between nascent branch junction to allow nucleation, but the precise role of
these two NPFs is rooted in the mechanism by which they stim- actin filaments during WASP-mediated activation remains a key
ulate the short-pitch conformation. While WDS proteins activate unresolved question.
through their interactions with the clamp, WASP binds to two sites In conclusion, the structure of activated Arp2/3 complex bound
far from the clamp6,8,11,28–30,35 (Supplementary Fig. 2). At these sites, to its nucleated actin filament provides a new structural context for
the Arp2/3-interacting segment in WASP, WASP-CA, engages the interpretation of over two decades of biophysical and biochemical
complex to stimulate the short-pitch conformation while the WASP studies, while also providing the foundation for future experiments
V region recruits actin monomers to the complex6,8,11,28–30,35,36. The aimed toward understanding how the highly conserved Arp2/3
activated structure presented here, along with structural and bio- complex nucleates actin filaments in response to different cellular
chemical data on WASP6,8,11,28–30,35, indicate that the WASP-C segment signals.

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Nature Structural & Molecular Biology Articles
1 Pointed 2 3
end
Clamp
Arp3 Dip1
Arp3 Dip1 Arp3
Twisted Dip1 Twisted Clamp
Twisted
twisting
C′
BEG
ARPC1
Arp2 Splayed Arp2 Arp2
Twisted Twisted Twisted

Barbed Short
end BEG pitch
Actin
(n)

Twisted Arp3
Flattening

Dip1 1
Dip 1
Arp3 Dip
Elongation
Arp3
int. Arp2 int.
Flattening
(n)
Arp2
Flat Arp2
C′
actin Actin Twisted C′
(n) monomer
Flat Actin
(n)
6 5 4 Twisted

Fig. 6 | Proposed mechanism of Arp2/3 complex activation by Dip1. In state 1, Arp2/3 complex is in an inactive state with the barbed end of Arp3
blocked by Arp2 and the barbed-end grooves (labeled BEG in state 2) are not properly arranged for interaction with actin. Dip1 binds to the clamp (red
dashed outline in state 1) to stimulate clamp twisting and movement of Arp2 into the short-pitch conformation (states 2 to 3). Arp2 in the short-pitch
conformation triggers partial flattening of Arp3, which opens up the Arp3 barbed-end groove for optimal long-pitch interactions with actin n (states 3
to 4). Steps 4 and 5 show that the actin monomer n flattens and stimulates flattening of Arp2 following binding to the complex; however, it is possible
that formation of the nucleus requires binding of additional actin monomers to the nascent filament. Dip1 binds tightly to activated Arp2/3 complex on
the pointed end of the actin filament17 (state 6), unlike WASP, which must be released for nucleation to proceed. Conformational states are indicated in
italicized text; int., intermediate (partially flattened) state of Arp3. Hatching indicates relative surface area buried at the short-pitch interface. For clarity,
ARPC3 is not depicted in this figure.

Online content 7. Rodnick-Smith, M., Liu, S.-L., Balzer, C. J., Luan, Q. & Nolen, B. J.
Any methods, additional references, Nature Research report- Identification of an ATP-controlled allosteric switch that controls
actin filament nucleation by Arp2/3 complex. Nat. Commun. 7,
ing summaries, source data, extended data, supplementary infor- 12226 (2016).
mation, acknowledgements, peer review information; details of 8. Rodnick-Smith, M., Luan, Q., Liu, S.-L. & Nolen, B. J. Role and structural
author contributions and competing interests; and statements of mechanism of WASP-triggered conformational changes in branched actin
data and code availability are available at https://doi.org/10.1038/ filament nucleation by Arp2/3 complex. Proc. Natl Acad. Sci. USA 113,
E3834–E3843 (2016).
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9. Xu, X.-P. et al. Three-dimensional reconstructions of Arp2/3
complex with bound nucleation promoting factors. EMBO J. 31,
Received: 14 April 2020; Accepted: 10 July 2020; 236–247 (2012).
Published: xx xx xxxx 10. Espinoza-Sanchez, S., Metskas, L. A., Chou, S. Z., Rhoades, E. &
Pollard, T. D. Conformational changes in Arp2/3 complex induced
by ATP, WASp-VCA and actin filaments. Proc. Natl Acad. Sci. USA 115,
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Methods third and seventh actin monomer addition steps. After the final addition of actin,
Protein purification. Rabbit skeletal muscle actin was extracted from acetone the reaction was incubated for 20 min before the addition of phalloidin to a final
powder (Pel Freeze, catalog no. 41995) by stirring in G-buffer (20 ml g–1, 2 mM concentration of 10 μM. For the inactive Arp2/3 complex sample, we attempted
Tris pH 8.0, 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2) on ice for 30 min to form coassemblies of Arp2/3 complex and Dip1 by mixing on ice 1.0 μM S.
before spinning in a Beckman JA-20 rotor at 16,000 r.p.m. for 30 min at 4 °C. pombe Arp2/3 complex and 3.0 μM Dip1 in a buffer containing 50 mM NaCl,
Supernatant containing actin monomers was filtered through glass wool. The 1 mM MgCl2, 1 mM ATP, 10 mM Tris pH 8.0 and 1 mM DTT. Only the Arp2/3
pellet was resuspended in the original volume of G-buffer and the extraction, complex per se, but no Dip1–Arp2/3 complex assemblies, was resolved by both
centrifugation and filtration steps were repeated. Actin was polymerized from two-dimensional (2D) and three-dimensional (3D) classification during cryo-EM
the combined supernatant by the addition of KCl to 50 mM and MgCl2 to 2 mM image processing.
final concentrations and stirred on ice for 1 h. Additional KCl was then added The same grid vitrification procedure was used for both samples: 4 μl of sample
to bring the final concentration of KCl to 0.8 M. After 30 min, the solution was was applied to a freshly glow-discharged 300-mesh UltrAuFoil R1.2/1.3 holey Gold
centrifuged for 2 h in a Ti70 rotor at 31,700 r.p.m. to pellet the polymerized grid (Quantifoil). Excess sample was manually blotted off with a dry Whatman
actin filaments. The pellet was gently washed with G-buffer and resuspended no. 1 filter paper for 5–7 s. Immediately after the blotting step, the sample
in G-buffer in a proportion of 3 ml g–1 of the starting stock of acetone powder. containing the grid was rapidly vitrified by plunge-freezing in liquid ethane at
Actin was then diluted to 5 mg ml–1 and dialyzed for 2 d with three changes of −179 °C. The entire cryo-grid preparation process was carried out in a cold room
G-buffer to depolymerize the actin filaments. The solution was then clarified at 4 °C and 98% relative humidity, to minimize excessive evaporation of sample
by centrifugation in a Ti90 rotor at 36,000 r.p.m. for 2 h, and actin was purified from the grid surface.
by size exclusion chromatography over a Sephacryl S-300 column equilibrated
with G-buffer containing 1 mM sodium azide. Fractions were stored on ice and Cryo-EM data acquisition. Cryo-EM data were acquired on a 200-kV Talos
used for experiments within 1 week. The concentration of actin was measured by Arctica transmission electron microscope equipped with a Falcon 3EC direct
absorbance using an extinction coefficient of 26,000 M−1 cm−1 at 290 nm. electron detector (Thermo Fisher). The automated data acquisition software EPU
Dip1 was purified as described previously3. Briefly, Dip1 was overexpressed as (Thermo Fisher) was used for data collection. For the Dip1-activated Arp2/3–actin
a glutathione S-transferase (GST) fusion in Escherichia coli using the pGV67-Dip1 complex, each micrograph was collected as dose-fractionated videos consisting
plasmid. Cells were homogenized and the lysate was clarified by centrifugation. of 45 fractions per video and at a nominal magnification of ×92,000. This
The clarified lysate was run over a Glutathione Sepharose affinity column and corresponded to a physical pixel size of 1.12 Å per pixel for each micrograph. The
eluted with reduced glutathione. The protein was then dialyzed overnight at 4 °C total exposure time per micrograph was 60 s and the total exposure was 36.35 e−/
with tobacco etch virus (TEV) protease then loaded onto a resource Q column in Å2 (0.81 e− per fraction). Data were collected underfocused with a nominal defocus
20 mM Tris pH 8.0, 50 mM NaCl and 1 mM DTT before elution with a 50–500-mM range between −1.00 and −1.75 µm (Extended Data Fig. 1a).
NaCl gradient. The protein was then concentrated and loaded onto a Superdex 200 Electron micrographs of the inactive Arp2/3 complex were collected using a
gel filtration column. The concentration of Dip1 was measured by absorbance previously described tilted data acquisition method18. Micrographs were acquired
using an extinction coefficient at 280 nm of 36,330 M−1 cm−1. by tilting the grids to angles of 20°, 30° and 40°. Additionally, data were collected
Schizosaccharomyces pombe Arp2/3 complex was purified as previously without tilting of the grids. All data were collected at a nominal magnification
described34. Briefly, S. pombe cells were lysed in a Microfluidics Model of ×120,000, corresponding to a size of 0.8757 Å per pixel, with nominal defocus
M-110EH-30 Microfluidizer and the lysate was clarified before the addition of ranging from −0.7 to −1.3 µm (Extended Data Fig. 1e). Each micrograph was
0.243 g ml–1 ammonium sulfate to the supernatant over 30 min with stirring. After collected as a dose-fractionated video consisting of 62 fractions over 40 s exposure
30 min, the pellet was resuspended in PKME (25 mM PIPES pH 6.8, 50 mM KCl, and a total exposure of 44.34 e–1/Å2 (0.72 e−1 per fraction).
1 mM EGTA, 3 mM MgCl2, 1 mM DTT and 0.1 mM ATP) then dialyzed overnight
against the same buffer before addition to a Glutathione Sepharose column charged Cryo-EM data processing. In total, 5,109 micrographs were collected for the
with GST-N-WASP-VCA. The Arp2/3 complex was eluted using a gradient of Dip1-activated Arp2/3–actin complex (Extended Data Fig. 1a). Beam-induced
PKME with a total of 150–1,000 mM KCl. Pooled fractions were then run on a motion correction and per-fraction radiation damage compensation over spatial
Mono Q column equilibrated in 10 mM PIPES pH 6.8, 25 mM NaCl, 0.25 mM frequencies (dose-weighting) were performed using the motion-correction
EGTA and 0.25 mM MgCl2 and eluted with a gradient of 25–500 mM NaCl. The algorithm implemented in the RELION v.3.0.6 suite (henceforth referred to as
dialysate was concentrated and loaded on a Superdex 200 gel filtration column RELION)38. Contrast transfer function (CTF) estimation for the micrographs
equilibrated with Tris pH 8.0, 50 mM NaCl and 1 mM DTT. The final concentration from aligned and summed fractions was estimated using the program Gctf39.
was determined by measuring absorbance at 290 nm using an extinction Based on the results for CTF estimation, we discarded 661 micrographs with
coefficient at 290 nm of 139,030 M−1 cm−1, and then the protein was aliquoted and poor confidence of fit or that were severely astigmatic. From the 4,448 remaining
flash-frozen. micrographs, particles were picked using the automated Laplacian Gaussian
His-tagged mouse capping protein (alpha-1, beta-2) in a pRSF Duet vector picking program implemented in RELION. This resulted in 3.5 million picked
(a gift from D. Kovar) was overexpressed in E. coli. Cells were lysed in 50 mM particles, which were extracted from micrographs using a square box size of
NaH2PO4 pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM imidazole and 10 mM 304 pixels and further binned to a box size of 76 pixels (voxel size of 4.48 Å per
β-mercaptoethanol with protease inhibitor tables. The lysate was run over a Talon pixel). These particles were subjected to multiple rounds of 2D classification. In
Metal Affinity resin (Clontech, no. 635501) and eluted with lysis buffer containing total, 2,346,578 particles belonging to 2D classes of the Arp2/3 complex capped
250 mM imidazole. Peak fractions were dialyzed overnight against 20 mM Tris pointed ends (Extended Data Fig. 1b) were selected for further processing. Using
pH 8.5, 50 mM NaCl, 5% glycerol, 0.01% NaN3 and 1 mM DTT, then loaded onto these particles, an ab initio 3D reference volume was created using cryoSPARC
a resource Q column, washed and eluted with a salt gradient of 50–500 mM NaCl. v.2 (ref. 40). This volume was used as initial reference for 3D processing. Multiple
Pure protein was dialyzed into storage buffer (20 mM HEPES, 1 mM EDTA pH .8.0, iterations of 3D refinements, followed by 3D classifications without alignment
200 mM KCl, 0.01% NaN3, 10% glycerol and 1 mM DTT) before flash-freezing. (clustering), were performed to isolate the best resolving 3D volume. Following
GST-Wsp1-VCA was overexpressed in E. coli, the cells lysed and the lysate this, 125,359 particles from 3D classes representing the Arp2/3 capped pointed
run over a Glutathione Sepharose column in 20 mM Tris pH 8.0, 140 mM NaCl, ends of actin filaments were then recentered and re-extracted as unbinned particles
2 mM EDTA and 2 mM DTT. The GST tag was cleaved on the column by the (304 pixels per box, 1.12 Å per pixel). Initial 3D refinement with this unbinned
addition of TEV protease and overnight incubation at 4 °C with gentle inversion. stack of particles resulted in a resolution map of 4.3 Å. Three-dimensional
The NaCl concentration was reduced to 100 mM by dilution and loaded on a binary masks for data processing were created using the 3D reference volume of
Mono Q column, washed and eluted with a gradient of 100–500 mM NaCl. Pooled the complex as template and expanding it by eight pixels, along with Gaussian
fractions were concentrated and loaded on a Superdex 75 gel filtration column. smoothing across ten pixels. For more accurate per-particle CTF estimation,
The concentration was measured using absorbance at 280 nm and an extinction CTF refinement within RELION was performed. Particles were also subjected to
coefficient of 5,690 M−1 cm−1 before flash-freezing. per-particle motion correction by Bayesian polishing41. These polished particles
were subjected to 3D refinement using a mask, resulting in reconstruction at 4.1-Å
Sample preparation for cryo-EM. To generate filaments capped on their pointed resolution. The 3D refined particle stack was subjected to three-class clustering. A
ends by the Dip1-bound Arp2/3 complex, we mixed 1.0 μM S. pombe Arp2/3 total of 110,433 particles belonging to the best 3D class were selected and subjected
complex, 5.0 μM Dip1, 1.0 μM Wsp1-VCA, 9.0 μM rabbit skeletal muscle actin to further 3D refinement. The final refined map was at a resolution of 3.9 Å (at
monomers and 0.6 μM mouse capping protein in a buffer containing 50 mM KCl, 0.143 Fourier shell correlation (FSC)) (Extended Data Fig. 3a). Because the cap
1 mM EGTA, 1 mM MgCl2, 0.2 mM ATP, 20 mM HEPES pH 7.5 and 1 mM DTT at and filament regions were flexible relative to each other, we decided to improve
room temperature. To keep the filaments short, we added all components except the quality of maps for these subregions by performing signal-subtraction-based
actin monomers and capping protein first, then spiked in one-third of the desired focused refinement42 (Extended Data Fig. 2a). Both subregions had the Arp2 and
final capping protein and actin monomers to 1.4 μM. After polymerization for Arp3 subunits in common. To rebuild the composite map, focused maps for the
20 min, we added additional actin monomers to bring the total actin concentration two regions were first fitted to a nonfocused map of the full complex, with the
to 2.3 μM. This process was repeated eight additional times to maintain a relatively Arp2 and Arp3 subunits as the overlapping subunits between the two maps. The
low concentration of unpolymerized actin monomers throughout the reaction. composite map was then generated by retaining the maximum valued voxel at
The remaining capping protein was added (one-third at a time) just before the each location from the two maps using the ‘vop maximum’ function within UCSF

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Articles Nature Structural & Molecular Biology
Chimera43 (Extended Data Figs. 2a and 1c,d). Local resolution of the map was (Extended Data Fig. 8), was defined by the following pairs of atoms: S. pombe
estimated using RELION’s local resolution estimation program (Extended Data Arp3: L179 Cα to R418 Cα; B. taurus Arp3: L163 Cα to R409 Cα; S. pombe Arp2:
Fig. 1c). The final map was sharpened by applying a B factor of –45.12 Å (ref. 2) S148 Cα to D383 Cα; B. taurus Arp2: L152 Cα to E387 Cα; O. cuniculus actin:
during the postprocessing step in RELION. T148 Cα to H371 Cα. Distance x4 measures the distance between the catalytic
The inactive Arp2/3 complex data were processed using CryoSPARC v.2.12.4 glutamine and the γ-phosphate (Fig. 4d). This distance was measured between
(ref. 40) (Extended Data Fig. 2b). A total of 5,309 micrographs, consisting of the γ-phosphate and the following residues: S. pombe Arp3, Q160; B. taurus Arp3,
videos, were collected with and without tilting and subjected to full-frame motion Q144; S. pombe Arp2, Q137; B. taurus Arp2, Q141; O. cuniculus actin, Q137. For
correction followed by patch-motion correction (Extended Data Fig. 1e). CTF distances x1, x2 and x3 the following structures were used: (1) Arp2 and Arp3 from
parameters were estimated locally using the patch CTF-estimation program. An all available structures of the Arp2/3 complex (PDB 1K8K, 1TYQ, 1U2V, 2P9I,
automated Gaussian-based blob picker was used to pick all the particles from 2P9K, 2P9L, 2P9N, 2P9P, 2P9S, 2P9U, 3DXK, 3DXM, 3DWL, 3RSE, 3UKU, 3UKR,
the micrographs, and 3,336,981 picked particles were then extracted from the 3ULE, 4JD2 and 6DEC (refs. 14,19,28,35,51–53), and the inactive and activated structures
micrographs using a square box of 368 pixels (0.8757 Å per pixel). These particles presented here). Because subdomains 1 and 2 of Arp2 are disordered in most of
were subjected to multiple rounds of 2D classification. After exclusion of particles these structures, fewer data points could be generated for the Arp2 subunit. (2)
belonging to classes having nonparticle features and aggregates, 450,747 particles Two representative actin monomer X-ray crystal structures in either the ADP- or
representing different views of the Arp2/3 complex (Extended Data Fig. 1f) were tATP-bound state (PDB 1NWK and PDB 2J6Z)54,55. (3) The highest-resolution
selected for 3D processing. These selected particles were then subjected to another cryo-EM structures of actin filaments with subunits in the ADP-, ADP-Pi- or
round of local-motion correction to more accurately correct per-particle drift. AMP-PNP-bound states15,16. In Fig. 4d, in addition to the activated structure
These particles were then subjected to three-class ab initio 3D reconstructions. In presented, distance x4 was measured in the following structures: PDB 4JD2, 2P9K,
total, 185,772 particles belonging to the intact inactive Arp2/3 complex class were 1TYQ, 3ULE, 3DWL, 2P9S, 2P9U, 1ATN, 1NWK, 2A40, 2A42, 2HBT, 6DJM
subjected to homogeneous refinement. To further separate heterogeneity, these and 5OOE.
particles were subjected to 3D classification by heterogenous refinement; 112,170 For analysis of the surface area buried at the interfaces in Extended Data Fig.
particles belonging to well-resolved, inactive Arp2/3 complex 3D class were finally 6c, coordinate files were processed using the Dockprep module in Chimera to add
refined using a static binary 3D mask, which resulted in a final reconstruction of missing side chains and were then submitted to the PISA protein interaction server
4.2 Å (at 0.143 FSC) (Extended Data Figs. 1g,h and 2b). Local resolution estimation (https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver)43,56. The clamp-twist
and sharpening of the map was performed in RELION using the two unfiltered angle for Fig. 5b was defined as either the dihedral between Arg32 Cα (ARPC4),
half maps (Extended Data Fig. 1f). The final map was sharpened by application of Ser 147 Cα (ARPC4), Ile262 Cα (ARPC2) and Arg18 Cα (ARPC2) in the S. pombe
a B factor of −116.219 Å (ref. 2). Arp2/3 complex or the homologous residues in the BtArp2/3 complex. The ARPC4
αD helix bend angle in Fig. 5c was defined using either Lys130 Cα, Asp143 Cα
Model building and analysis. The final maps generated from 3D reconstructions and Ser166 Cα in the SpArp2/3 complex or the homologous residues/atoms in the
mentioned above were used for atomic model building of each of the complexes. BtArp2/3 complex. In Fig. 2c the inactive Arp2/3 complex structure was overlaid
Structures of S. pombe inactive Arp2/3 complex lacking the Arp2 subunit (PDB on subdomains 1 and 2 of Arp3 in the activated structure to show movement of
3DWL) and F-actin subunits from rabbit (Oryctolagus cuniculus) ADP-actin Arp2. In Fig. 5a, the activated and inactive Arp2/3 complexes were superposed
filament (PDB 3J8I), were used as initial templates for atomic model building. using subdomains 1 and 2 of Arp3 and the globular portion of ARPC2. In Fig. 5d,
Ab initio computational models for the S. pombe Arp2 and Dip1 subunits were the αD helix of ARPC4 from each structure was superposed.
generated using the RaptorX44 (http://raptorx.uchicago.edu/StructPredV2/
predict/) and I-TASSER45 (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) Reporting Summary. Further information on research design is available in the
protein structure prediction servers. These models were initially rigid-body fitted Nature Research Reporting Summary linked to this article.
into the maps using the ‘fit map’ function in UCSF Chimera43. Flexible fitting
of the models into their respective maps was performed using the Namdinator Data availability
web server (https://namdinator.au.dk)36. Amino acid registers, backbone and EM maps and atomic models were deposited in the Electron Microscopy Data
side chain geometries for the models were manually fixed using Coot46. Density Bank and wwPDB, respectively, with accession entries EMD-21502 and PDB 6W17
corresponding to the ARPC3 subunit was poor, due to structural flexibility. The (Dip1–Arp2/3–actin) and EMD-21503 and PDB 6W18 (inactive Arp2/3 complex).
model for this region was truncated to Cβ and rigid-body fitted into the map. In
our active structure of Arp2/3 complex, we were able to build residues 235–366
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51. Nolen, B. J. & Pollard, T. D. Insights into the influence of nucleotides on actin to S.C., and by NIH grant nos. R01GM092917 and R35GM136319 to B.J.N. The SBU
family proteins from seven structures of Arp2/3 complex. Mol. Cell 26, cryo-EM center is supported by NIH grant no. S10 OD012272. M.S. was supported by
449–457 (2007). the Fulbright Association.
52. Nolen, B. J. & Pollard, T. D. Structure and biochemical properties of fission
yeast Arp2/3 complex lacking the Arp2 subunit. J. Biol. Chem. 283,
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53. Nolen, B. J. et al. Characterization of two classes of small molecule inhibitors
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were performed by M.S. and S.C. Atomic models were built by M.S. B.J.N performed
54. Otterbein, L. R., Graceffa, P. & Dominguez, R. The crystal structure of
structural analysis. All authors participated in manuscript preparation.
uncomplexed actin in the ADP state. Science 293, 708–711 (2001).
55. Graceffa, P. & Dominguez, R. Crystal structure of monomeric actin in the
ATP state. Structural basis of nucleotide-dependent actin dynamics. J. Biol. Competing interests
Chem. 278, 34172–34180 (2003). The authors declare no competing interests.
56. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from
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Additional information
Acknowledgements Extended data is available for this paper at https://doi.org/10.1038/s41594-020-0481-x.
Cryo-EM data were collected at the Stony Brook University (SBU) Cryo-EM center, Supplementary information is available for this paper at https://doi.org/10.1038/
and we thank G. Hu for providing support at this center. Computational infrastructures s41594-020-0481-x.
were provided by the cryo-EM and High-Performance Computing facilities at SBU. We Correspondence and requests for materials should be addressed to S.C. or B.J.N.
thank N. Samaroo and B. Ding for help with experiments, and acknowledge K. Prehoda
and members of the Nolen and Chowdhury laboratory for comments on the manuscript. Peer review information Peer review reports are available. Inês Chen was the primary
We also thank G. C. Lander for providing access to the Scripps Research cryo-EM editor on this article and managed its editorial process and peer review in collaboration
facility for determining the feasibility of the project. We thank D. Kovar for the with the rest of the editorial team.
capping protein expression vector. This work was supported by SBU start-up funds Reprints and permissions information is available at www.nature.com/reprints.

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Extended Data Fig. 1 | Cryo-Electron Microscopy of Arp2/3 complex in active and inactive states. a, A representative micrograph containing actin
filaments polymerized from Dip1-activated Arp2/3 complex. Circles mark the knob of density corresponding to Dip1-bound S. pombe Arp2/3 complex
at the pointed ends of filaments. Dip1 and Arp2/3 complex bind weakly to the ends of spontaneously nucleated actin filaments but strongly to Dip1–
Arp2/3–nucleated filaments34, so we concluded that most or all filaments with density for Arp2/3 complex were nucleated by Dip1-activated Arp2/3
complex. b, Reference-free 2D class averages obtained from particles corresponding to the knob shaped densities highlighted by circles in a. These class
averages show Dip1–Arp2/3–actin-filament complex adopts different orientations in vitrified ice. c, Two different views of the reconstructed 3D map of
active Arp2/3 complex bound to Dip1 and pointed end of nucleated actin filament, colored based on local resolution values of each voxel. d, Euler angle
distribution plot for particles contributing to the reconstructed map in c (map colored in gray at the center). e, A micrograph of S. pombe Arp2/3 complex
sample collected by tilting the stage to 40˚ showing uniform distribution of particles in ice. f, Representative reference free 2D class averages of Arp2/3
complex showing different orientations of the complex obtained from particles extracted from tilted and non-tilted micrographs. g, Two different rotated
views of the 3D reconstruction of Arp2/3 complex in an inactive state colored based on per-voxel local resolution values. h, Plot showing the Euler angle
distribution of particles that went into the final reconstructed inactive Arp2/3 complex map (colored in gray). Scale bars (white line) for the micrographs
in a and e represent distance of 25 nm and for the 2D class averages b and f represent 12.5 nm distance.

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Extended Data Fig. 2 | See next page for caption.

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Articles Nature Structural & Molecular Biology
Extended Data Fig. 2 | Single-particle cryo-EM data processing workflow for 3D reconstructions of activated and inactive Arp2/3 complex. a, Data
processing schematic for Dip1–Arp2/3–actin-filament complex. Particle stack was initially binned by factor of four and subjected to multiple rounds
of 2D and 3D classification to discard particles that did not correspond to the complex. The cleaned particle stack was re-extracted without binning
and further subjected to downstream 3D processing. All 3D classifications were performed without image alignment (referred to as clustering). Final
composite map was generated by combining focused maps corresponding to the Dip1-Arp2/3 cap (Region-1, colored purple) and the Arp-actin filament
(Region-2, colored blue). b, Schematic for processing of Arp2/3 complex (inactive state) cryo-EM data. Particles extracted from all micrographs (tilted
and non-tilted) were cleaned by multiple rounds of 2D classification and then subjected to multiple ab initio 3D reconstructions. Particles corresponding to
intact Arp2/3 complex were further subjected to downstream data processing that lead to a 4.2 Å 3D reconstruction of Arp2/3 complex in inactive state.

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Extended Data Fig. 3 | Reconstructed maps and atomic models of subunits. Fourier shell correlation (FSC) plots between masked (yellow) and unmasked
(orange) half maps, and final map and model (blue) for global resolution estimates for a, Dip1–Arp2/3–actin-filament complex, and b, inactive Arp2/3
complex. EM density for each subunit is shown as gray mesh. c, Arp3, d, Arp2, e, ARPC1, f, ARPC2, g, ARPC3, h, ARPC4, i, ARPC5, j, Dip1, and k, actin
maps with built models. l, Maps and models corresponding to the long helices from the ‘clamp’ subunits, ARPC2 (cyan) and ARPC4 (blue) in the inactive
state (left) and active state (right).

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Extended Data Fig. 4 | The structure of inactive S. pombe Arp2/3 complex is nearly identical to other inactive Arp2/3 complex structures. a, Structural
superposition of inactive S. pombe Arp2/3 complex (colored subunits) from the cryo-EM reconstruction presented here with inactive Bos taurus Arp2/3
complex (semi-transparent gray subunits, PDB 4JD218). Structures were superposed using Arp3 subdomains 1 and 2, Arp2, ARPC1, ARPC2 and ARPC4.
b, Structural superposition showing that Arp3 from the S. pombe inactive Arp2/3 complex structure shows a more open nucleotide binding cleft than other
ATP-bound Arp3 structures. Subdomains 1 and 2 of inactive S. pombe Arp3 (orange) were superposed on inactive BtArp3 (transparent gray, PDB 4JD2).
Cyan arrow shows the direction of rotation of subdomains 3 and 4 toward subdomains 1 and 2 during cleft closure. Red arrow shows the axis of rotation of
cleft closure. A rotation of 10.3° about this axis is required to close the S. pombe nucleotide cleft to the same extent as the BtArp2/3 structure. The open
cleft in the inactive S. pombe Arp2/3 complex is the major structural difference between the two structures.

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Extended Data Fig. 5 | Comparison of Arp2 and Arp3 in the activated complex to actin in the nucleated filament. a, Superposition of Arp3 Cα atoms
from the active structure to actin subunit n in the nucleated filament. The RMSD between 256 pruned atom pairs is 1.31 Å (across all 354 pairs: 3.45). b,
Superposition of Arp2 Cα atoms from the active structure to actin subunit n in the nucleated filament. The RMSD between 314 pruned atom pairs is 0.92 Å
(across all 352 pairs: 2.74).

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Extended Data Fig. 6 | Partial flattening allows Arp3 to make short pitch interactions that mimic those of a short pitch actin dimer. a, Left, Arp3 from the
active (orange) and inactive (semi-transparent gray) Arp2/3 complex structures, with definition of measurements in plot. Top, the twist angle (𝜑) is the
dihedral angle between the centers of mass (blue spheres) of each of the four subdomains (see Methods). Bottom, distance x1 between Y92 Cα and K235
Cα in S. pombe Arp3. Right, plot of the twist (𝜑) versus distance x1 in inactive or active structures of actin, Arp2 or Arp3. Data points highlighted in yellow
are from the activated Arp2/3 complex structure. A subset of structures are labeled: em (from cryo-EM reconstructions here); PDB 6DEC (Spin90 bound
to Bos taurus (Bt)Arp2/3 complex, chain ID in parentheses21); PDB 4JD2 (BtArp2/3 complex bound to GMF18); PDB 1NWK and PDB 1J6Z (representative
actin monomer structures bound to ATP or ADP, respectively53,54). b, Superposition of inactive (twisted) Arp3 from the inactive S. pombe Arp2/3 complex
cryo-EM structure and Arp3 from the activated structure overlaid using subdomains 3 and 4. Arp2 in the short pitch conformation from the activated
structure is shown in pink. Partial flattening of Arp3 upon activation allows subdomains 1 and 2 to make closer contacts with Arp2 in the short pitch
conformation. c, Surface area buried at the short pitch interface between Arp2 and Arp3 in the active structure (model 1) or theoretical models in which
the Arps are modeled in the short pitch conformation and either Arp2 (model 2), Arp3 (model 3), or both (model 4) adopt the twisted conformation.
Models 2, 3 and 4 were constructed by individually superposing either Arp2, Arp3, or both subunits from the inactive structure onto their corresponding
subunit in the activated structure using Cα atoms from subdomains 3 and 4. d, Comparison of the short pitch interface between Arp3 and Arp2 (left
panel) or actin (n) and actin (n + 2) right panel. The 9° rotation of Arp2 brings the helix harboring E231 from Arp2 closer to subdomain 1 of Arp3, allowing
E231 to interact with R418.

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Extended Data Fig. 7 | Uncurling of the W-loop facilitates long-pitch interactions with actin. a, Close up of long pitch interactions between the Arp3
barbed end groove (BEG) and the actin D-loop. Activated Arp3 (orange) is overlaid on an inactive (transparent) structure using subdomains 3 and 4. A
structure of Bos taurus Arp2/3 complex (PDB 4JD2) was used as the inactive structure in this and all analyses in this figure as some sidechains at the
barbed end of Arp3 are missing in the inactive S. pombe Arp2/3 complex structure presented here. b, Long pitch interactions between the Arp2 BEG in the
activated (pink) or inactive (transparent) structure (PDB 4JD2). Arp2 from each structure was superposed using subdomains 3 and 4. c, Structure of Arp3
from activated Arp2/3 complex showing the definitions of the measurements made in panel d. d, Plot of the twist angle (𝜑) versus distance x2—which
measures uncurling of the W loop—for inactive or active structures of actin, Arp2 or Arp3. em: structures from the cryo-EM reconstructions presented
here; PDB 6DEC: structure of Spin90 bound to Bos taurus (Bt)Arp2/3 complex26; PDB 4JD2: BtArp2/3 complex bound to GMF18; PDB 1NWK and PDB 1J6Z:
representative actin monomer structures bound to ATP or ADP, respectively53,54.

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Extended Data Fig. 8 | Flattening causes changes in the barbed end groove that facilitate long pitch interactions with actin subunits. a, Molecular
surface representation of active Arp3 showing perspective of close up views of the barbed end grooves (BEGs) depicted in b. b, BEG of inactive (PDB
4JD2) or active Arp2 or Arp3 with engaged D-loop from actin subunit n + 1 or n. Residue numbers are for the S. pombe Arp2/3 complex. Green spheres and
dashed line show distance x3, which measures the distance between the C-terminus and the front of the BEG. c, Plot of the twist angle versus distance x3
for inactive or active structures of actin, Arp2 or Arp3. Individually labeled data points are as described in Extended Data Fig. 7d.

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Extended Data Fig. 9 | Close up of nucleotide clefts of Arp2 and Arp3. a, Stereo image of the nucleotide binding cleft of Arp3 showing the density of the
modeled ATP phosphates and conserved catalytic residue Q160. PG: gamma phosphate. b, Stereo image of the nucleotide binding cleft of Arp2 showing
the density of the modeled ATP phosphates and conserved catalytic residue Q137. c, Stereo image of Arp2 from the active structure (pink) superposed
with actin (cyan) from the cryo-EM structure of AMP-PNP bound actin filaments PDB 6DJM15 showing the nucleotide and the nucleotide binding cleft.

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Extended Data Fig. 10 | The clamp subunits twist during activation of Arp2/3 complex. a, Model-based surface representation of ARPC2 and ARPC4
in active or inactive structure (PDB 4JD2) with residues contacting Arp2 or Arp3 (within 3.7 Å) colored magenta or orange, respectively. The Bos taurus
inactive structure (PDB 4JD2) was used for the inactive structure in this analysis because the inactive S. pombe Arp2/3 complex structure presented here
is missing some sidechains at the interface with the clamp. b, Overlay of ARPC2 and ARPC4 from the active structure on ARPC2 and ARPC4 from the
inactive S. pombe Arp2/3 complex structure, respectively. Residues 1-271 of ARPC2 and 4-140 of ARPC4 were used to overlay the structures.

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