Structural basis for scaffolding-mediated assembly
and maturation of a dsDNA virus
Dong-Hua Chena, Matthew L. Bakera, Corey F. Hryca, Frank DiMaiob, Joanita Jakanaa, Weimin Wuc,1,
Matthew Doughertya, Cameron Haase-Pettingelld, Michael F. Schmida, Wen Jiangc, David Bakerb,
Jonathan A. Kingd, and Wah Chiua,2
aNational Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, TX 77030;
West Lafayette, IN 47907; and
bDepartment of Biochemistry, University of Washington, Seattle, WA 98195;
dDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
cDepartment of Biological Sciences, Purdue University,
Edited by David DeRosier, Brandeis University, Waltham, MA, and approved December 1, 2010 (received for review October 22, 2010)
Formation of many dsDNA viruses begins with the assembly of
a procapsid, containing scaffolding proteins and a multisubunit
portal but lacking DNA, which matures into an infectious virion.
This process, conserved among dsDNA viruses such as herpes
viruses and bacteriophages, is key to forming infectious virions.
Bacteriophage P22 has served as a model system for this study
in the past several decades. However, how capsid assembly is
initiated, where and how scaffolding proteins bind to coat proteins
in the procapsid, and the conformational changes upon capsid
maturation still remain elusive. Here, we report Cα backbone
models for the P22 procapsid and infectious virion derived from
electron cryomicroscopy density maps determined at 3.8- and
4.0-Å resolution, respectively, and the first procapsid structure at
subnanometer resolution without imposing symmetry. The procap-
sid structures show the scaffolding protein interacting electrosta-
tically with the N terminus (N arm) of the coat protein through its
C-terminal helix-loop-helix motif, as well as unexpected interac-
tions between 10 scaffolding proteins and the 12-fold portal
located at a unique vertex. These suggest a critical role for the scaf-
folding proteins both in initiating the capsid assembly at the portal
vertex and propagating its growth on a T ¼ 7 icosahedral lattice.
Comparison of the procapsid and the virion backbone models
reveals coordinated and complex conformational changes. These
structural observations allow us to propose a more detailed mole-
cular mechanism for the scaffolding-mediated capsid assembly
initiation including portal incorporation, release of scaffolding pro-
teins upon DNA packaging, and maturation into infectious virions.
cryo-EM ∣ asymmetric reconstruction ∣ P22 phage ∣ asymmetric procapsid ∣
cursor (procapsid) to an infectious virion (1–4). In addition to the
coat proteins, the procapsid requires scaffolding proteins, absent
from the virion, for proper assembly, and a portal for DNA
packaging and subsequent DNA ejection. However, despite a
half-century of research on icosahedral viruses, it remains unclear
how initially identical subunits adopt both hexameric and penta-
meric conformations in the virus and select the correct locations
needed to form closed shells of the proper size (5). Packaging of
DNA through the portal is accompanied by the exit of scaffolding
proteins from the procapsid and conformational changes in the
coat proteins as the capsid matures (2, 6).
Understanding the molecular mechanisms of dsDNA virus
assembly and maturation requires knowledge of the interactions
among the coat, scaffolding, and portal proteins, all of which are
essential for these processes. X-ray crystallography (7–9) and
electron cryomicroscopy (cryo-EM) (10–12) have yielded near-
atomic to atomic resolution models of several dsDNA icosahe-
dral viruses and provided a structural framework of interactions
among their coat proteins. However, the structural details of
procapsid portal incorporation, scaffolding protein binding and
sDNA viruses infecting both prokaryotes and eukaryotes
share a common assembly pathway proceeding from a pre-
release, and capsid shell expansion during maturation remain
largely unknown. In particular, the scaffolding-dependent asso-
ciation of the coat protein with the portal ring at a single fivefold
capsid vertex has been difficult to characterize. The structures
reported here of the P22 virions and procapsids reveal the con-
formational transition upon maturation, as well as the contacts
among the portal, scaffolding subunits, and the coat subunits.
These contacts offer a mechanism by which icosahedral shell
assembly is initiated and controlled in P22 and possibly in some
other dsDNA viruses.
Results and Discussion
Cryo-EM Structure of P22 Procapsid at 3.8-Å Resolution. The bacter-
iophage P22 procapsid is a well-characterized morphogenetic
precursor of the mature phage, consisting of hundreds of copies
of the gene 5 (gp5) coat and gene 8 (gp8) scaffolding proteins,
multiple copies of three ejection proteins (gp7, gp16, gp20, also
known as pilot proteins), and a unique multisubunit gene 1 (gp1)
portal (13). We used single-particle cryo-EM to determine its
structure at 3.8-Å resolution, enforcing icosahedral symmetry
with approximately 23,400 particle images (Fig. 1A and Fig. S1 A
and B). The procapsids were isolated from cells infected with
nonsense mutants of the terminase complex defective in DNA
packaging and represent the physiological precursor prior to
DNA packaging and capsid maturation.
From our map, Cα backbone models for each of the seven
structurally similar but nonidentical copies of the coat protein
(gp5) in the asymmetric unit of the T ¼ 7 procapsid were built
with a hybrid de novo modeling method using Gorgon (14) and
Rosetta (15) (Fig. 1 B and C and Fig. S2 A, C and E). The coat
proteins are organized as pentamers and skewed hexamers as
Author contributions: D.-H.C., J.A.K., and W.C. designed research; D.-H.C., M.L.B., C.F.H.,
F.D., J.J., and W.W. performed research; D.-H.C., M.L.B., C.F.H., F.D., C.H.-P., and D.B.
contributed new reagents/analytic tools; D.-H.C., M.L.B., C.F.H., F.D., M.D., J.A.K., and
W.C. analyzed data; and D.-H.C., M.L.B., C.F.H., F.D., M.F.S., W.J., J.A.K., and W.C. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: The icosahedral cryo-EM density maps of bacteriophage P22 procapsid,
empty procapsid, and virion have been deposited in the Electron Microscopy data bank,
European Bioinformatics Institute, http://www.ebi.ac.uk/ (accession nos. EMD-1824,
EMD-1825, and EMD-1826, respectively). The asymmetric density map of bacteriophage
P22 procapsid and the 12-fold averaged portal have been deposited in the Electron
Microscopy data bank (accession nos. EMD-1827 and EMD-1828, respectively). The Cα
models for the asymmetric units of the bacteriophage P22 procapsid and virion have been
deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2XYY and 2XYZ,
1Present address: Laboratory of Structural Biology Research, National Institute of Arthritis
and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1015739108 PNAS ∣ January 25, 2011 ∣ vol. 108 ∣ no. 4 ∣ 1355–1360
previously reported for the GuHCl treated procapsid (16–20).
Each of the gp5 models consists of eight distinct domains: A do-
main, extra-density (ED) domain, E loop, D loop, F loop, P do-
main, N arm, and a long helix (Fig. 1B). No significant sequence
homology to other proteins is found for the entire coat protein.
Part of the gp5 has similar secondary structure elements and a
characteristic coat protein fold, which is found in HK97 and other
phages (7, 8, 11, 19, 21, 22). However, the fold of the ED domain
is different from the previous model based on a lower resolution
cryo-EM map (20).
Bacteriophages T4 (23), Lambda (24), and ϵ15 (10) utilize
additional coat proteins that bind to the outside of the major coat
protein, which stabilizes the virion. In bacteriophage HK97 (7),
no such additional proteins are present in the mature capsid.
Whereas P22 has only one coat protein, the coat protein contains
the ED domain at the capsid surface (Fig. 1B). This domain has a
unique fold, composed primarily of β-strands, a short α-helix, and
a protruding loop (the “D loop”) (Fig. 1B and Fig. S2 A and C).
The D loopis located at the intersubunit interface between neigh-
boring capsomeres, and provides strong interactions across the
local twofold axes (Fig. S3A). Out of 21 temperature-sensitive
mutations that affect the coat protein monomer folding and/or
capsid assembly (25–27), 14 are found in this domain, suggesting
it plays a key role in stabilizing the monomeric coat protein and/
or the procapsid shell (Fig. S3C). Previous intermediate-resolu-
tion studies proposed that this domain stabilizes the coat protein
monomer (20). Our model suggests that more extensive interac-
tions in the D loop and ED domain are involved in stabilizing
the entire procapsid (see Fig. 4 below) in lieu of the covalent
modifications exhibited by HK97 phage or the additional coat
proteins in T4, Lambda, and ϵ15 phages.
Scaffolding Protein and Its Interactions with Coat Protein. V-shaped
densities at the hexamers (Fig. 1D), which cannot be accounted
for by the coat protein model, are found on the inner surface
of the coat protein shell (Movie S1). These densities are not
observed in a separate 7.0-Å reconstruction of the scaffolding
protein-deficient P22 procapsid (empty procapsid) (Fig. S4A),
suggesting that these V-shaped densities are part of the scaffold-
ing proteins. Each V-shaped density agrees in size and shape
with the NMR structure of the P22 scaffolding protein C-terminal
region (residues 238–303, Protein Data Bank ID 2GP8) consist-
ing of a helix-loop-helix motif (28) (Fig. S4B). Although all of
the V-shaped densities within an asymmetric unit are similar, the
interface between each scaffolding protein density and the coat
protein varies in the skewed hexamer.
Earlier studies suggested that there are two distinct scaffolding
binding sites on the coat protein (29, 30). Based on our model,
one of the two scaffolding binding sites occurs at the negatively
charged residues on the N arm of the coat protein (Fig. 1 E
and F), which interact with the positively charged C-terminal
domain of the scaffolding protein (28). These electrostatic inter-
actions are consistent with previous biochemical studies (31, 32).
Note that the interactions between the scaffolding protein and
coat protein have never been visualized in such fine detail and
modeled in any authentic bacteriophage procapsid, although pre-
vious attempts at lower resolutions were made in herpes virus
capsid (33) and P22 (17).
The second scaffolding protein binding site is located at the tip
of the A domain of the coat protein, which is bent toward the
interior of the procapsid shell (Fig. 1E and Fig. S4B). In four
(S1, S2, S4, S5) of the six positions at each skewed hexamer, the
scaffolding protein spans two coat proteins, binding to the N arm
of one coat protein and the A-domain tip of an adjacent coat
protein. In the other two positions (S3, S6) at each hexamer, the
scaffolding protein does not contact either its own or the adjacent
coat protein’s A-domain tip; the scaffolding protein is only bound
to one coat protein at its N arm. At the pentamers, the scaffolding
density appears considerably weaker with the base of the V shape
of the scaffolding protein situated below the A-domain tips of two
adjacent coat proteins (Fig. 1D).
typical cryo-EM image of the P22 procapsid. (B) A Cα backbone model from residues 10–425 with eight annotated domains for one gp5 subunit derived from
the density. The model is colored from blue (N terminus) to red (C terminus). (C) The Cα model for the entire procapsid. (D) The internal surface view of a portion
of procapsid density map along an icosahedral threefold axis (labeled “3”), showing the V-shaped densities in red around hexamers (labeled “6”) and pen-
tamers (labeled “5”). The V-shaped densities at pentamers, shown in a black circle, are displayed at a lower contour level than the rest of the map. The density
map was low-pass filtered from 3.8 to 7.0 Å, and was radially colored according to the color scheme shown at the bottom except the inset of a pentamer in
the black circle using different color scheme shown on the right. (E) Interactions between the coat protein (colored ribbons, N arm colored in blue) and the
C-terminal helix-loop-helix motif of the scaffolding protein (red cylinders) in a hexamer. (F) The charge distribution of one procapsid subunit model.
The scaffolding protein-binding region (dashed circle) is negatively charged.
The icosahedral structure of the P22 procapsid at 3.8-Å resolution and the interactions between the scaffolding protein and the coat protein. (A) A
www.pnas.org/cgi/doi/10.1073/pnas.1015739108Chen et al.
Based on our cryo-EM structures of P22 procapsids, which
were averaged from approximately 23,400 particle images, every
coat protein is associated with one scaffolding protein. As the
scaffolding protein densities at the pentamers appear at a lower
isosurface value, it may be possible that there is only partial
occupancy of scaffolding protein at the pentamers. Regardless,
this number of scaffolding proteins is likely higher than 250–300
copies as previously deduced from biochemical data (34, 35).
Interactions Between Scaffolding Proteins and the Unique Portal in
Asymmetric Procapsid Structure. To investigate the interactions
between the scaffolding proteins and the portal vertex, the pro-
capsid was reconstructed to 8.7-Å resolution without imposing
icosahedral symmetry (Fig. 2 A–C, Fig. S5, and Movie S2). This
map shows the density features and locations of the coat protein
and the scaffolding proteins to be the same as those in the ico-
sahedral map (Fig. 1D), thus validating the proposed interaction
sites between the scaffolding and coat proteins (Fig. 1E). The
map also reveals a unique portal at one of the twelve vertices,
as well as its interactions with the capsid shell. Similar to the
biochemically isolated portal (36, 37), the in situ portal of the
procapsid has 12-fold symmetry and can be divided into the clip,
stem, wing, and crown domains (36, 38) (Fig. S5C). An extra
barrel-shaped domain extending next to the crown domain in the
portal toward the interior of the capsid has not been previously
reported (Fig. S5C), and may correspond to the C-terminal por-
tion of the portal protein as in P-SSP7 phage (39). The clip do-
main is loosely connected to the coat proteins by the P domains
and F loops from the surrounding five hexamers (Fig. S5 B–F).
As in the icosahedral reconstruction, the C termini of the scaf-
folding proteins are also observed on the inner surface of capsid
shell in the asymmetric map (Fig. 2C). Again, their densities are
nonequivalent in the skewed hexamer, truly representing unique
interactions between the coat and scaffolding proteins as shown
above (Fig. 1 D and E). Unexpectedly, a set of scaffolding sub-
units is interposed between and interacts with the portal and coat
subunits (Fig. 2 C and D). The interaction of the scaffolding pro-
tein with the portal is actually much more extensive than that of
the coat protein with the portal, because the “loop” in the helix-
loop-helix motif of the scaffolding protein is in close contact with
the wing domain of the portal (Fig. 2D and Movie S2). The pre-
sence of the scaffolding proteins here explains why the portal is
not incorporated into procapsids in the absence of scaffolding
proteins (40) and the mutation (G287E) in the loop, proximal
to the portal, of the helix-loop-helix motif in the C-terminal por-
tion of scaffolding protein influences the portal incorporation
into procapsids (41).
Our asymmetric map shows that the C termini of 10 scaffolding
proteins (Fig. 2C) interact with the portal in P22, which is
different from a recent model for ϕ29 phage (42). Nonidentical
interactions between the wing domain of the portal and the
surrounding scaffolding proteins may be a structural mechanism
to accommodate the symmetry mismatch between the 12 portal
proteins and the 10 scaffolding proteins at the portal vertex. It
should be emphasized that the 10 numbered scaffolding proteins
(Fig. 2C) are not associated with a pentamer (there is no penta-
mer at this vertex). Rather, each of the five surrounding hexamers
contributes two of its associated scaffolding proteins to the inter-
action with the portal. Each of these two scaffolding proteins has
different orientations with respect to the portal, in addition to the
symmetry mismatch mentioned above (Movie S2).
In contrast to the well-ordered C termini bound to the coat
proteins, the rest of the scaffolding proteins appear as two disor-
dered concentric shells in the icosahedral and asymmetric
(Fig. 2B) reconstructions. These rings presumably arise from
the hundreds of elongated scaffolding subunits with their 10∶1
axial ratio (43, 44). The inability to visualize the conformations
regions of the scaffolding subunits are not ordered or vary from
particle to particle. This region of the scaffolding protein may
function to exclude cytoplasmic proteins from entering the grow-
ing shell, in addition to interacting with the ejection and portal
proteins and self-associating during procapsid assembly.
Virion Structure at 4-Å Resolution and Conformational Changes upon
Capsid Maturation. In contrast to the spherical procapsid, the P22
virion appears more angular and has the characteristic DNA
fingerprint (Fig. 3A). A 4.0-Å icosahedral reconstruction was
obtained from approximately 18,300 virion particle images
(Fig. S1 C and D). The skewed hexamers in the procapsid become
more symmetric in the virion, with smaller openings at the center
of the hexamers (Fig. 1C, Fig. 3B, and Fig. S3 A and B). These
changes are coordinated with the increase in diameter from ∼610
to ∼710 Å and thinning of the shell wall in the virion (Fig. S1 A
and C and Movie S3). Cα backbone models were again built for
the seven coat proteins using our hybrid de novo method (Fig. 3B
and Fig. S2 B, D, and F). Our models of the procapsid and virion
differ from earlier models based on lower resolution maps of
empty and expanded procapsids (20) and reveal structural
changes not previously seen.
Despite having similar folds, conformational differences are
observed in the various domains of the coat protein in the procap-
sid and virion (Fig. 3 C and D and Movie S3). A set of conforma-
tional changes in the coat protein occurs in the two previously
mentioned regions that interact with the scaffolding protein.
procapsid map at 8.7-Å resolution. (B) A central slice of the asymmetric re-
construction. The inner two disordered concentric shells (red) are composed
mainly of scaffolding proteins. The portal (gray) interacts with the scaffold-
ing proteins at positions pointed by black arrows and with the coat proteins
at the positions marked with circles. In (A), the portal density (gray) was
segmented out and 12-fold averaged. In both A and B, the density map
was color-coded using the same scheme as in Fig. 1D, except the portal is
in gray. (C) The C termini of 10 scaffolding proteins are labeled from 1 to
10, from five hexamers (circled) surrounding the portal vertex. The view di-
rection is at the level of, and normal to, the dashed line in B and the portal
has been removed for clarity. (D) Side view of the interaction model among
the 10 scaffolding protein C termini (red cylinders), coat proteins (ribbons),
and the 12-fold averaged portal density (gray). A difference map between
the procapsid asymmetric structure and its icosahedrally averaged structure
showed no significant difference for the bound scaffolding proteins and cap-
sid shell; the only difference was at the portal. The red cylinders were fit into
the scaffolding protein densities of icosahedrally averaged structure.
Asymmetric reconstruction of the P22 procapsid. (A) The asymmetric
Chen et al. PNAS
January 25, 2011
The A-domain tip (Fig. 3 E and F) flexes toward the centers of
the capsomeres during maturation, narrowing the capsomere
openings and resulting in a more symmetric hexamer (Fig. S3 A
and B). The N arm, another site for scaffolding protein binding, is
rotated approximately 20° clockwise in the virion, placing it
below the β-sheet region of the P domain (Fig. 3 E and F). In
addition, the long helix is “unkinked.”
Conformational changes in regions of the coat protein not
directly interacting with the scaffolding protein also occur during
capsid expansion and maturation. In the procapsid, the tip of the
E loop (G60–A67) interacts with the tip of a P domain (G396–
S402) of a coat protein in an adjacent asymmetric unit across the
In the virion, the E loop is shifted approximately 20° outward
(Fig. 3D), resting on the large β-sheet region of the P domain in
the tips of the three P domains remain in the same position at the
In the procapsid, several charged residues at the tip of the D
loop extend into a pocket of complementary charges formed by
part of the ED domain and β-sheet portion of the P domain of the
neighboring coat protein subunit in an adjacent capsomere along
the local twofold symmetry axes (Fig. 4A). Seven temperature-
sensitive mutations that affect coat protein monomer folding
and/or capsid assembly (25–27) are located within this binding
pocket or the loop itself (Fig. S3C). Along with the adjacent F
loops, these interactions stabilize neighboring capsomeres in the
procapsid (Fig. 4A). In the virion, these interactions do not exist;
rather the D loops are shifted out of their pockets in the adjacent
asymmetric unit. To maintain stability in the virion, this shift of
the D loops forms new electrostatic interactions between the two
D loops of the subunits across the capsomeres (Fig. 4B). Thus,
intercapsomere interfaces in the procapsid are composed of the
D and E loops interacting with the P domains as well as the F
loops, whereas only interactions between the D loops seem to be
involved in intercapsomere interfaces in the virion.
During capsid expansion, intracapsomere interactions com-
pensate for the loss of intercapsomere interactions in the virion
(Fig. S3 A and B). Helix 2 and helix 3 in the A domains (Fig. S2 C
and D) of neighboring subunits form strong lateral electrostatic
interactions that vary considerably between the procapsid and
virion. In the procapsid, the interacting surface area at this inter-
face is similar for the pentameric subunits and four of the six
hexameric subunits (labeled as C1, C2, C4, and C5 in Fig. S3A).
In the other two hexameric procapsid subunits (labeled as C3
and C6 in Fig. S3A), there is a marked reduction (∼10%) in total
interaction surface area at this interface. Helix 2 and helix 3 in
these two subunits, due to the skewing, are shifted such that the
electrostatic interactions between the neighboring subunits are
out-of-register and do not participate in any interactions. As
the skewed hexamers become more equivalent in the virion, these
two helices move into register in all hexameric positions. The
interacting surface area is nearly identical at all hexameric and
pentameric positions, forming the core intracapsomere interac-
tions. Along with the E loop/P domain interactions in the virion
(Fig. S3 A and B), these interactions likely account for the in-
creased stability of the virion but, at the same time, still accom-
modate the skewing of the procapsid hexamers.
Molecular Mechanism for Capsid Assembly, Scaffolding Protein
Release, and Capsid Maturation. Based on our cryo-EM structures
and their associated models, we can propose a detailed molecular
mechanism for P22 procapsid assembly, including portal incor-
poration, scaffolding protein release, and capsid maturation
(Fig. 5). Beyond P22, this model also may help elucidate the con-
formational switching involved in forming hexamers and penta-
mers, as well as the cascade of events associated with packaging
the viral genome in icosahedral viruses.
To form a functional P22 procapsid, at least four types of pro-
teins are required: coat, scaffolding, ejection, and portal proteins,
in which the portal proteins form a unique 12-fold portal complex
at a fivefold vertex. The lack of scaffolding proteins results in
the failure to incorporate the portal and can lead to incomplete
particles (29, 32, 40, 45). Based on our density maps, we propose
that the formation of a unique portal complex with the requisite
scaffolding and coat proteins is likely the key for initiating proper
procapsid assembly (Fig. 5 A and B). Scaffolding proteins may
play a critical role during the capsid assembly nucleation because
the portal would not be incorporated into the procapsid when the
scaffolding proteins are absent. Note that the scaffolding protein
in bacteriophage HK97 is a domain at the N terminus of the coat
protein, and hexamers and pentamers are present in solution
in vitro (46). In contrast, in bacteriophage P22 the scaffolding
conformational changes upon maturation. (A) A typical cryo-EM image of
P22 virion. (B) The Cα backbone model of gp5 (residues 1–425) for the virion.
Outside (C) and tangential (D) views of a subunit model from the virion
(magenta) and procapsid (cyan) superimposed, showing conformational
changes at the long helix, D loop, and E loop. An interior view of a procapsid
subunit (E) and of a virion subunit (F). The movement of A-domain tip
(orange) and the N arm (navy) are apparent in the virus maturation.
The icosahedral structure of the P22 virion at 4.0-Å resolution and
view of local twofold interactions in the procapsid (A) and the virion (B)
Structural difference at the capsomere interface. (A and B) Zoom-in
www.pnas.org/cgi/doi/10.1073/pnas.1015739108 Chen et al.
protein is a separate protein from the coat protein and no evi-
dence exists for preformed hexamers or pentamers in solution
(43, 44, 47, 48), suggesting that capsomeres form during P22 pro-
capsid assembly. Once nucleated, procapsid assembly proceeds
by the addition of scaffolding and coat subunits to the growing
shell until the full procapsid is assembled with the proper size
and shape as directed by the scaffolding proteins (Fig. 5 C and D).
Coat proteins are likely added as monomers or dimers, with the
mediation of scaffolding proteins, because our structures show
that each scaffolding protein’s C-terminal helix-loop-helix motif
interacts with the N arm of the corresponding coat protein.
Though the exact timing is not known, ejection proteins are also
incorporated and possibly interact with the scaffolding and portal
proteins before procapsid assembly is completed (29, 49). The
ejection proteins are likely located close to the portal but remain
to be identified in our asymmetric procapsid map.
The control of conformational switching involved in forming
hexamers and pentamers at the correct locations in icosahedral
viruses remains poorly defined. In P22, the fact that four of
the scaffolding subunits bind to two different coat protein subu-
nits in a skewed hexamer (Fig. S4B) may explain how scaffolding
proteins control the ratio between hexamers and pentamers
during icosahedral shell assembly. In the absence of scaffolding
proteins, the coat proteins sometimes polymerize into smaller
T ¼ 4 shells (45). Both the T ¼ 4 and T ¼ 7 particles have 12
pentamers, though the T ¼ 4 particles have 30 skewed hexamers
compared to 60 in T ¼ 7 particles. In the presence of both pro-
teins, the binding of scaffolding proteins to coat proteins shifts
the equilibrium towards hexamers, generating more authentic
T ¼ 7 particles. Presumably, the skewing of the hexamers in the
procapsid is also a result of those two subunits being tilted out of
the plane of the capsomere (labeled as C3 and C6 in Fig. S3A).
This observation, along with the differences in the coat-scaffold-
ing protein interactions in a procapsid asymmetric unit, may
dictate the capsid geometry and the interfaces between capsome-
res. Although scaffolding proteins mediate the assembly of the
procapsid shell, they are not required for maintaining the capsid
shell, as evident in our empty procapsid structure (Fig. S4A) and
the T ¼ 4 capsid structure (45), both of which show the skewed
conformation for hexamers.
Once the capsid shell forms (Fig. 5D), DNA packaging can
begin. In the early stages of DNA packaging, the terminase
complex (gp2 and gp3) docks against the portal and hydrolyzes
ATP to drive DNA into the procapsid shell through the portal.
Upon entry into the procapsid shell via the portal channel, DNA
then encounters the scaffolding proteins, which have negatively
charged residues at their N termini (35). We propose that, as
more negatively charged DNA enters, the scaffolding subunits
are expelled out of the shell through the large central openings
(∼18 × 42 Å) of the skewed hexamers (Fig. 5E) (the openings at
pentamers in the procapsid seem to be closed because they are
similar in size to the ones in pentamers and hexamers of the vir-
ion) and the capsid is triggered to expand by ∼100 Å. The fold of
the coat subunit remains basically unchanged, though numerous
structural changes, notably the unkinking of the long helix, the N
arm rotation, the flexing of the A-domain tip, and movements of
D loop and E loop, occur (Fig. 3 C–F). Capsid expansion does not
appear to be directed by the scaffolding protein, as the icosahe-
dral structures of the procapsid shell with and without scaffolding
protein bound appear to be virtually identical (Fig. S4A). Rather,
the coat protein itself may exist in several metastable states
(including those found in the procapsid and virion) and, depend-
ing on the state of the capsid shell, may be able to switch confor-
mations to best accommodate local interactions necessary for
maintaining the integrity of the capsid. Theforces behind matura-
tion, i.e., the dsDNA packaging which results in the release of the
scaffolding proteins, likely drives the coat protein into alternative
contacts with neighboring coat proteins and necessitates small
shifts in the coat protein structure. In the P22 procapsid, the
portal may also be involved in controlling capsid expansion, as
seen in phage T4 procapsids (50). In our procapsid asymmetric
structure, the scaffolding proteins were observed to extensively
connect to the portal. Once the scaffolding proteins are expelled
by the packaging DNA, the removal of these connections
between the portal and the scaffolding proteins could possibly
trigger the rearrangement of coat proteins during DNA packa-
ging. To accommodate such rearrangements while still encapsu-
lating the viral genome and providing a stable structure,
secondary coat proteins and/or additional domains are required
to maintain capsid stability and potentially propagate structural
transitions as suggested previously (20, 36, 37). Only after the
capsid has fully expanded and packaged its DNA, the tail hub
proteins (gene 4 and gene 10), along with the needle (gene 26)
and tail spikes (gene 9) are attached to form an infectious
virion (Fig. 5F).
In short, our cryo-EM studies of P22, in two morphogenetic
states, provide structural details and support a more developed
and detailed mechanism for capsid nucleation, scaffolding-
mediated assembly, scaffolding protein release, and DNA-trig-
gered maturation. As there is conservation among dsDNA
viruses, a similar mechanism is likely to be found in other dsDNA
viruses, including those that infect animals, such as adenoviruses
protein release and capsid maturation. (A and B)
The portal (gray) associates with scaffolding (red)
and coat (cyan) proteins to initiate procapsid assem-
bly. The assembly continues with the addition of scaf-
folding and coat subunits to the growing shell (C)
until the full procapsid is assembled (D). DNA is then
packaged into the procapsid shell through the chan-
nel of the portal by the terminase motor. The scaf-
folding proteins are released by the electrostatic
forces from DNA being packaged and exit through
the central large openings of hexamers (E). During
the release of scaffolding proteins, conformational
changes associated with the maturation transition
occur. After capsid expansion and DNA packaging,
the tail hub, needle, and tail spikes are attached
to the portal to form an infectious virion (magenta)
(F). In B and C, the insets show the side views. In D
and F, the insets show one hexamer and the adjacent
pentamer rotated from the side view to the end-on
Pathway for capsid assembly, scaffolding
Chen et al.PNAS
January 25, 2011
Materials and Methods Download full-text
P22 procapsids and infectious virions were purified from Salmonella typhi-
murium using an established procedure (47, 51). The samples were applied
to Quantifoil grids, flash-frozen using a Vitrobot and imaged. Data for the
icosahedral and asymmetric reconstructions were collected on photographic
film on a 300-kV JEM-3000SFF with a specimen temperature of ∼4 K. Addi-
tional images for the asymmetric procapsid reconstruction were captured
on a Gatan 10 × 10 k CCD (US10000XP, model 990) with 2× binning on a
300-kV JEM-3200FSC with energy slit of 20 eV in the in-column energy filter.
Data processing for the icosahedral procapsid and virion structures from
approximately 23,400 and 18,300 particle images, respectively, was per-
formed using EMAN (52) with newly implemented programs for global
optimization and defocus compensation. The asymmetric procapsid recon-
struction was obtained with approximately 43,850 particle images as
previously described (39). Resolutions for the icosahedral structures were
assessed using the 0.5 criterion of the Fourier shell correlation (53) from
two independent half datasets. The resolution for the asymmetric procapsid
reconstruction was assessed based on the comparison with the 3.8-Å procap-
sid icosahedral reconstruction. Segmentation of the coat protein subunits
was done using Chimera (54). De novo Cα backbone modeling for each of
the seven subunits in an asymmetric units in both procapsid and virion
was performed using a hybrid of modeling tools and cryo-EM constrained
refinement available in Gorgon (14) and Rosetta (15, 55), respectively.
Iterative model refinement in Coot (56) and Gorgon was used to optimize
the models and remove clashes of subunits within and across capsomeres.
ACKNOWLEDGMENTS. We thank Xiangan Liu for his advice on the asymmetric
reconstruction and Juan Chang and Frazer Rixon for discussions and assis-
tance in the manuscript preparation. This research was supported by the
National Institutes of Health (R01AI0175208, P41RR002250, R01GM079429,
and PN2EY016525), the National Science Foundation (NSF IIS-0705644 and
IIS-0705474), and The Robert Welch Foundation (Q1242). This work was sup-
ported in part by the access to the computing resources from Rice University
Computational Research Cluster supported by NSF (CNS-0421109), National
Energy Research Scientific Center supported by the Department of Energy
(DE-AC02-05CH11232), and the Texas Advanced Computing Center at the
University of Texas at Austin (MCB100112).
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