Chapter 15

Structural and Mechanical Characterization of Viruses

with AFM
Álvaro Ortega-Esteban, Natália Martı́n-González,
Francisco Moreno-Madrid, Aida Llauró, Mercedes Hernando-Pérez,
Cármen San Martı́n, and Pedro J. de Pablo

Abstract
Microscopes are used to characterize small objects with the help of probes that interact with the specimen,
such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy
(AFM) the probe is a nanometric tip located at the end of a micro cantilever which palpates the specimen
under study as a blind person manages a walking stick. In this way AFM allows obtaining nanometric
resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also
enables not only the manipulation of single protein cages, but also the characterization of every physico-
chemical property able of inducing any measurable mechanical perturbation to the microcantilever that
holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we
describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic
methodologies devoted for extracting physical information, such as mechanical and electrostatic properties.
We also explain how a convenient combination of AFM and fluorescence methodologies entails monitoring
genome release from individual viral shells during mechanical unpacking.

Key words Atomic force microscopy, Force curve, Nanoindentation, Beam deflection, Tip, Cantile-
ver, Stylus, Topography, Aqueous solution, Disruption, Breaking, Fatigue, Electrostatics

1 Introduction

A protein cage can be roughly stated as any closed structure built

out of protein subunits that defines an internal cavity at the nano-
meter scale. Although viruses illustrate at most the definition of
protein cages, nonviral structures, such as Bacterial Microcompart-
ments (BMCs) [1], vault particles [2], and artificial virus-like struc-
tures [3–5], can also be included in this description. The basic
architecture of a virus consists of the capsid, a shell made up of
repeating protein subunits (capsomers), packing within the viral
genome [6]. Far from being static structures, viruses are highly
dynamic nucleoprotein complexes that transport and deliver their

Nuno C. Santos and Filomena A. Carvalho (eds.), Atomic Force Microscopy: Methods and Protocols, Methods in Molecular Biology,
vol. 1886, https://doi.org/10.1007/978-1-4939-8894-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2019

259
260 Álvaro Ortega-Esteban et al.

genome from host to host in a fully automatic process. Viral parti-

cles are endorsed with specific physicochemical properties which
confer to their structures certain metastability whose modulation
permits fulfilling each task of the viral cycle at the right time. These
natural designed capabilities have impelled using viral capsids as
protein containers of artificial cargoes (drugs, polymers, enzymes,
minerals) [7] with applications in biomedical and materials sciences.
Both natural and artificial protein cages have to protect their cargo
against a variety of physicochemical aggressive environments,
including molecular impacts in highly crowded media [8], thermal
and chemical stresses [9], and osmotic shocks [10]. Thus, it is
important to use methodologies that supply information about
protein cages stability not only under different environments, but
also its evolution upon structural changes. In this vein, structural
biology techniques such as electron microscopy (EM) and X-ray are
used to unveil the structure-function interplay, revealing high-
resolution impressive structures of protein cages [11]. However,
these methodologies require a heavy average of millions of particles
present in the crystal (X-ray) or thousands of structures for the
model reconstruction (cryo-EM). Thus they provide limited infor-
mation on possible structural differences between individual parti-
cles in the population that distinguish them from the average
structure. In addition, these approaches require conditions (vac-
uum) far away of those where protein shells are functional (liquid).
Thus, these techniques preclude the characterization of protein
shells dynamics and properties in real time. Indeed, the advent of
single molecule technologies has demonstrated that mechanical
properties of biological molecular aggregates are essential to their
function [12]. It is evident that the exploration of these properties
would complete the structural biology methodologies (EM and
X-ray) to find the structure-function-property interplay of protein
cages. Atomic force microscopy (AFM) may not only characterize
the structure of individual protein particles in liquid milieu, but also
to obtain physicochemical properties of each particle, such as
mechanics or electrostatics. In addition, the nano-dissectional abil-
ities of AFM allow the local manipulation of protein shells to learn
about their assembly/disassembly. In this chapter, we pretend to
give a general overview of how to apply AFM methods to protein
shells. Our tour starts with a basic review of the recipes for attach-
ing protein cages to solid surfaces and continues describing the
most successful modes for imaging protein shells with AFM so far.
Subsequently we describe the nanoindentation methodology,
which probes the stiffness, breaking force, brittleness, etc., of indi-
vidual protein shells. Afterward we focus on the effects of mechani-
cal fatigue on individual particles, and how to access to the cargo to
probe it properties, sometimes in combination with fluorescence
microscopy. In the last part, we focus on the methodologies to
measure the electrostatics of individual protein particles.
 AFM of Protein Shells 261

2 The Attachment of Protein Shells to Solid Surfaces

As with every specimen to be examined with AFM, immobilization

of protein shells to a flat solid substrate is a sine qua non-requisite.
Viral shells are typically attached to substrates by using physical
interactions of viral shells with the substrate, including polar, non-
polar, and van der Waals interactions [13]. Physisorption traps
protein cages on the surface without creating chemical bonds that
might alter their structure. Each type of protein shell has indivi-
dualized features such as hydrophobic patches or local charge den-
sities [14] that can be used for adsorption, via hydrophobic and/or
electrostatic interactions on different substrates, such as glass, mica,
and HOPG (Highly Oriented Pyrolytic Graphite) (Fig. 1a). Mica
and HOPG surfaces are layered materials whose preparation con-
sists of removing the topmost layer with adhesive tape, exposing a
fresh surface ready for experiments. HOPG presents a nonpolar
surface and protein cages adsorb via hydrophobic interactions
[15]. Sometimes this interaction is very strong and can destroy
the samples, although this is not usually the case [15, 16]. The
hydrophobic interaction can be reduced when functionalizing glass
surfaces with hexamethyldisilazane (HDMS) [17]. Mica exhibits a
negatively charged surface that can be changed to positive by using
counterions [13]. This allows the attachment of protein containers
through negatively charged locations. Let us focus on the specific
case of human adenovirus particles (HAdv). In this case about
20 μL of virus solution presenting 1.5–2.0
1012 particles/mL
was incubated on the surface and washed with the corresponding
buffer after 20 min. Afterward the liquid cell is formed between the
cantilever holder and the sample (Fig. 1b). Figure 1c shows that
HOPG collapses some HAdv particles, indicating a strong nonpo-
lar (hydrophobic) interaction. However, silanized glass reduces the
attachment force and allows imaging intact icosahedral particles
exhibiting fivefold, twofold and threefold symmetry orientations
on the surface (see Fig. 1c, d). Interestingly, using 150 mM NiCl2
on mica [13] induces the adsorption of HAdv particles with three-
fold symmetry orientation; thus, protein particles exhibit a triangu-
lar facet (Fig. 1d). Adsorption of protein cages on surfaces may also
induce a reduction of the particle height. For instance, the same
P22 bacteriophage structure undergoes a larger collapse on HOPG
than on silanized glass (Fig. 2), revealing that the interaction with
HOPG is stronger. This variety of adsorption phenomena may
change for each type of protein shell, since different structures
expose different residues in the external layer, thus requiring differ-
ent adsorption methodology. From a practical point of view pre-
dictions on proteins shells adsorption are difficult to make, and one
uses the “trial and error” methodology to find the best conditions.
262 Álvaro Ortega-Esteban et al.

Fig. 1 Attaching protein shells on surfaces. (a) HOPG, glass, and mica bare
substrates before attaching the viruses. (b) Illustration of the experimental
system. Protein cages (red dots) and cantilever are not to scale. To visualize
the real scale, please visit https://www.youtube.com/watch?v¼MZb8C0f7Kdg.
(c) hAdV on HOPG, glass, and mica. D) Individual hAdV particles showing twofold,
threefold, and fivefold symmetry orientations after adsorption on the surface.
(Adapted from Ortega-Esteban’s thesis)

3 Imaging Methodology

Typically, in AFM the tip scans the sample in x, y, and z directions by

using piezo actuators. While x and y scanners move in a preestab-
lished way over a square region, the cantilever bends following the
surface topography. Either the cantilever deflects perpendicularly to
the surface applying a normal force (Fn) (Fig. 3a), or it bends
laterally by torsion exerting a dragging force parallel to the surface
 AFM of Protein Shells 263

Fig. 2 Protein shells deforms on the surface. (a) P22 bacteriophage particles on glass and HOPG oriented at
fivefold and 3/2 symmetry axes. (b) Comparison of topographical profiles obtained on two particles adsorbed
on glass (dark) and HOPG (red) obtained from (a). (c) Comparison of average height of particles adsorbed at
different orientations and substrates. (Adapted from [16])

Fig. 3 AFM working modes. (a, b) show the normal and lateral force concepts, respectively. (c, d) indicate
contact and jumping modes, respectively. (Adpated from Ortega-Esteban’s thesis)

(Fl) (Fig. 3b). Both Fn and Fl are monitored by focusing a laser

beam at the end of the cantilever, whose reflection is registered in a
four quadrant photodiode. Thus, each pixel of the image located at
a particular position of planar coordinates (x, y), will be associated
264 Álvaro Ortega-Esteban et al.

with certain bending values of the cantilever Fn and Fl. If the

specimen is not strongly attached or if it is too soft, it can be
swept or modified under large bending forces. To avoid this effect
as much as possible, a feedback loop is engaged to Fn to move the
z piezo position in such way that Fn is kept constant. In this
operational approach, termed as contact mode (Fig. 3c), the AFM
topography map will have x, y, and z coordinates. The torsional
bending force Fl of the cantilever exerts about 40 times the flexural
bending force Fn [18]. These dragging forces are not problematic
with solid surfaces, such as mica [19] or specimens which are held
secure by their neighbors, such as purple membrane [20] or similar
periodical structures. However, isolated specimens, such as DNA
molecules (2 nm in diameter), are especially prone for being dam-
aged in contact mode [21]. Individual protein cages are also sus-
ceptible to undesired modifications by lateral forces. Their size of
tens of nanometers offers a large topographical aspect ratio that is
difficult to track for the feedback loop. A typical approach for
surpassing this limitation is the use of fixation agents, such as
glutaraldehyde. In such conditions AFM provides images whose
resolution is comparable to that of some EM images [22]. Never-
theless, since glutaraldehyde structurally reinforces the specimens
[23, 24], it precludes any characterization of processes or proper-
ties of intact native viruses, such as disassembly or mechanical
properties [25]. Other approach includes developing imaging
modes that avoid dragging forces as much as possible. In jumping
mode (JM), also called pulse force mode [26, 27], the lateral tip
displacement occurs when the tip and sample are not in mechanical
contact, thereby avoiding to a large extent shear forces (Fig. 3d).
JM performs consecutive approach-release cycles at every pixel of
the sample. In each cycle, known as force vs. distance z curve (FZ,
Fig. 4a), z-piezo approaches tip and sample from noncontact (label
1 at Fig. 4a) until establishing mechanical contact (label 2 at
Fig. 4a) and reaching certain feedback force (label 3 at Fig. 4a).
After a few milliseconds, the z-piezo retracts about 100 nm until
releasing the tip from the surface (label 4 at Fig. 4a) [27]. Subse-
quently the scanner moves laterally to the next pixel, and the
process starts again. When operating in air (Fig. 4a, left), forward
and backward curves are similar after releasing the surface and the
feedback force (Fig. 4a, right) is always above cantilever deflection.
Interestingly, the AFM cantilever experiences a viscous drag while
moving up and down in liquid, giving rise to a hysteresis loop
(Fig. 4b, left). As the cantilever approaches the surface the dragging
force produces a spurious deflection that hides the tip-sample con-
tact point (Fig. 4b, right). If the dragging deflection equals the
feedback set point force, then the z piezo retracts before tip-sample
contact. This imposes the feedback set point force to be higher than
the viscous drag (Fig. 4b, left). This viscous hysteresis can be
removed in jumping mode plus [28] from the curve (Fig. 4c,
 AFM of Protein Shells 265

Fig. 4 Force curves used for imaging in JM. (a) Illustrates the normal force signal
during a FZ in air condition (left) and the corresponding illustration (right)
indicating the set point of the maximum Fn. (b) As Fig. (a) but in water. (c)
Corrected FZ after subtracting the viscous force (Adapted from [28])

left), allowing the use of set point feedback forces close to the
cantilever thermal noise, i.e., ~100 pN (Fig. 4c, right). Although
other AFM dynamic modes are also able to image protein shells in
liquid conditions, it is difficult to control the applied force [29].

4 Tip-Sample Dilation

The typical radius of the tip apex for usual cantilevers (OMCL-
RC800PSA) is ~20 nm, and it is comparable to the protein cages
size. In this case tip size plays an important role on image resolution
by inducing a lateral expansion, termed dilation, of the imaged
specimen [30]. The WSxM software implements a geometrical
dilation algorithm that allows the simulation of the dilation of the
structure of a protein shell. For instance, by using Chimera [31] it is
possible to access to a protein shell structure, such as the electron
microscopy model. By using the Surface Color option by height, it
is possible to generate a gray-scale image to capture the topography
variation in a given orientation. The TIFF format of this image can
be imported by WSxM software and calibrated. The dilation algo-
rithm asks for the tip radius, and the dilated structure is calculated.
266 Álvaro Ortega-Esteban et al.

Fig. 5 Dilation effects in the protein shell of bacteriophage phi29. (a) Represents
the AFM image of a phi29 prohead. In (b) the bright color illustrates an EM
structural model of phi29, and the dark area indicates the dilation corresponding
to a tip of 10 nm in diameter. The illustration in (c) indicates the dilation as a
function of the tip size: dark, red and blue curves are the topographical profiles
obtained with tips of 0.5 nm, 5 and 10 radius in diameter, respectively

Figure 5 exemplifies the dilation of the phi29 bacteriophage pro-

head EM structure [32]. Figure 5a shows the real AFM image of a
phi29 prohead vertically oriented, and Fig. 5b illustrates the dila-
tion effect in the dark structure, compared with the actual size
profiled inside in bright color. The dilation strongly depends on
the tip size, as shown in Fig. 5c.

5 The Mechanics of Protein Shells

The mechanical properties of protein shells have been the subject of

intense research during the last years. AFM was initially applied to
study virus shells [17, 33]. In this vein, virus mechanics have been
 AFM of Protein Shells 267

related to packed genome [34–38], maturation [24, 39–42], artifi-

cial cargo [16], structural modulation [43], etc. [44]. Protein shells
of nonviral origin have been also investigated, such as vault particles
[15] and encapsulin [45]. Here we will focus on the methodology
for extracting the mechanical information of individual protein
nanocages with AFM.

5.1 Nanoindentation The first method consists of monitoring the indentation of the
AFM tip into individual protein cages. Single force versus distance
curve (FZ) experiments are performed by pushing on the top of a
selected protein shell (Fig. 6a). The particle is continuously
zoomed in by reducing the x–y scanning size until the bump of
the very top is under the whole scan (~50
50 nm2) and y scan is
switched off. Afterward the FZ is executed on the particle at a
typical speed of 50 nm/s to allow the water leaving the virus
when it is squeezed [46]. After the contact between tip and particle
is stablished, FZ typically shows an approximate linear behavior,
which corresponds to the elastic regime of the shell and ascribes to

Fig. 6 Single indentation assay. (a) Cartoon showing the three main stages during nanoindentation experiment
on a protein cage: before contact (1), during deformation (2) and after breaking (3). (b) Evolution of Fn along the
z-piezo elongation. Forward curve exhibits the three stages commented in (a). (c) AFM topographies of
adenovirus before and after nanoindentation. (d) Nanoindentation data extracted from (c), showing the shell
deformation. Inset compares topographical profiles of (b) (before and after breakage in blue and red,
respectively) showing a crack of ~20 nm in depth
268 Álvaro Ortega-Esteban et al.

the mixed bending of the cantilever and sample deformation

(Fig. 6b, label 2). By controlling the z-piezo elongation it is possi-
ble to go back and forth several times, and the particle elastically
deforms in a reversible way. When the z-piezo elongation surpasses
the critical indentation, particle breaks (Fig. 6c) inducing a variety
of peaks in the FZ, that resemble the chaotic penetration of the tip
apex trough the cage (Fig. 6b, 3). Afterward FZ is linear again and
represents the cantilever bending. By performing a FZ on the
substrate, and assuming that it is much more rigid that the cantile-
ver, we can obtain the cantilever deformation (Fig. 6b, solid line).
The subtraction of sample from substrate curves allows isolating the
deformation of the cage (Fig. 6d). From this graph we can obtain a
few mechanical parameters, such as the stiffness or spring constant,
by fitting the elastic part from 0 to 8 nm (k ¼ 0.18 N/m); the
breaking or yield force, which is the force value when the elastic
regime finish at 8 nm (Fb ¼ 1.4 nN), and the critical indentation δc,
which is the deformation of the virus when it breaks (8 nm). Thin
shell theory relates the protein shell stiffness with the Young’s
2
modulus as k  E hR , where h is the thickness of the shell and R its
radius. [47]. The area enclosed between forward and backward
curves from indentation 0 up to 8 nm is the energy used to break
the cage. In this case, it is about 8 nm
nN, i.e., 8
1018 J or
~2000 kBT, which approaches the order of magnitude of the total
energy used for assembling all the proteins [48]. In addition, the
critical strain εc ¼ δc/h, where h is the initial height of the protein
cage as measured with AFM, reflects the brittleness or the mechan-
ical stability of protein cages [16]. In this case, εc ¼ 8/60 ¼ 0.13.
This value implies that this particle deforms about 13% without
breaking. The analysis of the chaotic part of the indentation curve
sometimes provides further information. For instance, in vaults
particles it was associated with the individual proteins unzipping
while the particle was being broken during the nanoindentation
experiment [15]. While most protein cages break showing fragile
fracture [42], sometimes plastic fracture is also present [49]. The
nanoindentation methodology also allows the probing of the self-
recovery abilities of protein cages after breakage. For instance, vault
particles demonstrated self-healing capabilities able of removing
the cracks produced after indentation [15]. The precise control of
nanoindentation permits the access to the inner cargo of protein
cages. For instance, the consecutive application of nanoindentation
cycles in human adenovirus cracks open the shell in a controlled
fashion to probe the mechanical properties of the core [50]. These
mechanical properties relate to the condensation state of dsDNA.

5.2 Molecular The breaking force describes the maximal force survivable by the
Fatigue shell and collapse the particle by inducing large and uncontrollable
and Disassembly changes in its structure. It is thus difficult to derive consequences
about disassembly, since in the cycle of many virus shells, for
 AFM of Protein Shells 269

instance, disassembly takes place by losing individual capsomers in

an ordered manner [6]. A protein cage must also resist a constant
barrage of sublethal collisions in crowded environments [51]. Equi-
partition theorem provides an estimation of the energy transferred
in a molecular collision to be ~ 32 kB T , which is far below that the
energy supplied by single indentation assay experiments, as we
explained before. Imaging of individual protein shells with AFM
in jumping mode requires thousands of load cycles (FZs) at low
force (~100 pN per pixel, Fig. 4c) [28]. A rough estimation indi-
cates that ~10kBT is transferred to the particle at every cycle [52],
very close to the molecular collisions value. The continuous imag-
ing of a particle enables the evaluation of any structural alteration
while subjected to cycle load at low forces. In this vein, molecular
fatigue experiments have demonstrated to be a disassembly agent
able of recapitulating the natural pathway of adenovirus uncoating
[28]. Therefore molecular fatigue provide additional mechanical
information by reporting on shell stability against such multiple
deformation cycles at low force (~100 pN) [41], well below the
breaking force (Fig. 6d). Let us exemplify the molecular fatigue
methodology in the case study of lambda bacteriophage [52] which
infects E. coli. Upon maturation, cementing protein gpD adds to
hydrophobic patches at the external surface of the expanded shell
[53]. In the case of E. coli living in the gut, mature particles must
survive outside the bacteria in an environment which is ~7 times
more viscous that the host milieu. Therefore, lambda particles
should resist about seven times more molecular collisions outside
than inside the bacteria. Molecular fatigue offers an excellent work-
bench for probing the resistance of undecorated and decorated
particles by mimicking molecular impacts. The experiment consists
of continuously imaging individuals of each structure and monitor-
ing the appearance of the first damage (Fig. 7). The label of each
topography image (Fig. 7a) indicates the times that it has been
scanned from being untouched to the final collapse. The average
loading cycles needed for first damage on 7 and 8 undecorated and
decorated particles, respectively, are depicted in Fig. 7b. It is worth
noting that a force of 120 pN was used on decorated particles,
because 100 pN was not enough to induce any damage [52]. We
can conclude that decoration with external proteins increases the
resistance of protein cages against molecular impacts in crowded
media.

6 AFM/Fluorescence Combination

The association of AFM with other techniques, such as mass spec-

trometry, has shown to be a valuable tool for understanding the
biophysics of protein cages [38]. Here we discuss the methodology
for studying the mechanical unpacking of protein shells by
270 Álvaro Ortega-Esteban et al.

Fig. 7 Mechanical fatigue of lambda bacteriophage shells. (a) AFM topography images of undecorated and
decorated particles, showing intact, damaged and collapsed states. Labels indicate the number of images
obtained on the same particle. (b) Average number of cycles applied to induce the first damage on decorated
and undecorated particles (Adapted from [52])
 AFM of Protein Shells 271

combining AFM and total internal reflection fluorescence micros-

copy (TIRFM). Many viruses disassemble at the right place and
time to release their genome. Specifically, human adenoviruses
(hAdV) disassemble in a stepwise manner through the cytoplasm.
Afterward hAdV particles reach the nucleus and release the dsDNA
through the nuclear pore. HAdV packs inside 35 kbps of dsDNA
and 25 MDa of condensing proteins, forming the core. The pro-
teins of this viral chromatin are cleaved by the viral protease during
maturation. Previous AFM uncoating experiments [41] indicated
that the immature core remained visible as a condensed blob,
whereas the core of the mature virus could not be resolved. These
results already suggested a difference in the core organization and
might be important in the genome diffusion after the virus shell
disassembly. dsDNA strands do not necessarily attach to the surface
after diffusing out of the broken cage, and it is difficult to monitor
this process with AFM. However, fluorescence microscopy allows
exploring the diffusion of non-immobilized biomolecules, such as
diffusing dsDNA. AFM is used to crack open single viral capsids.
The genome exposure can be tracked with a DNA-specific inter-
calating fluorescent dye (YOYO-1) that can only access the DNA
after the capsid has been opened (Fig. 8a). The integration of a
single molecule fluorescence microscope with AFM require the
monitoring of the fluorescence signal at the surface to avoid not
only the background signal of the AFM probe itself, but also the
light coming from the bulk solution. By using TIRFM, the tip apex
and cantilever remain largely out of the evanescent excitation field
(~100 nm) [54]. Figure 8b presents simultaneous AFM and fluo-
rescence images of a single hAdV particle before and after nanoin-
dentation. Fluorescence image shows emission only after the
particle is broken. Figure 8c shows simultaneous FZ and fluores-
cence signals while the particle is broken: fluorescence emission
starts right after the particle is broken. Statistics of mature and
immature particles data illustrates (Fig. 8d) the evolution of fluo-
rescence emission after capsid breakage. Fluorescence spots are
larger for mature particles, demonstrating that the structural
change of the core during maturation unwinds DNA and making
it more accessible to the fluorophore [55]. This genome expansion
might help in its diffusion through the nuclear pore from mature
particles. This new approach paves the way to study the diffusion of
artificial cargos packed within protein cages for technological
purposes [56].

7 Electrostatic Characterization

Electrostatics is one of the fundamental driving forces of the inter-

action between biomolecules in solution. The recognition events
between viruses and host cells are dominated by both specific and
272 Álvaro Ortega-Esteban et al.

Fig. 8 AFM/fluorescence combination for monitoring mechanical unpacking. (a) Sketch of AFM/fluorescence
combination for monitoring the access of YOYO-1 to released DNA. (b) AFM and fluorescence data of a hAdV
particle before and after releasing DNA. (c) Simultaneous force (orange) and fluorescence (green) data during a
nanoindentation experiment that disrupts the particle and release DNA. (d) Evolution of the fluorescence signal
along time after particles disruption for mature (blue) and immature (green) particles (Adapted from [55])

nonspecific interactions. Electrostatic force is a nonspecific interac-

tion which is determined by the charge of viral particles. Detection
of electrostatics in liquid environments with AFM was already
developed for surfaces in early 90s [57]. More recently the electro-
statics of single biomolecules, such as dsDNA [58], and the avidin-
streptavidin system [59] have been explored. Although there are a
variety of AFM dynamic methods to measure electrostatic charge
[60, 61] we will focus on the simplest methodology based on a
single indentation assay. It is convenient to take a careful look at the
contact between the tip and sample in Fig. 6d. We see that this
contact does not show a sharp kink but it is a little curved. The
 AFM of Protein Shells 273

origin of this curvature is the repulsive electrostatic force between

the protein cage and the Si3N4 tip, both negatively charged. In
liquid environment and beyond pure mechanics, there is a plethora
of long and short-range forces that affect the interaction between
an AFM tip and the protein cage [62]. In particular, when the tip
approaches the protein cage, the interaction force before contact
can be described in the frame of Derjaguin-Landau-Verwey-Over-
beek model (DLVO) [63], which accounts for the electrostatic Fe
an van der Waals FvdW forces. The interaction between two planes is
expressed by:
2σ s σ t λz Ha
F DLVO ¼ F el þ F vdW ¼ eD 
ε0 ε 6πz 3
where σ s and σ t are the charge density of sample and tip, respec-
tively; ε0ε is the dielectric constant times the permittivity of vac-
uum; z is the tip-sample distance, Ha is the Hammaker constant of
the tip-sample system, and λD is the Debye length that determines
the range of the electrostatic forces (double Debye layer). Debye
Length depends solely on the salt concentration c as λD ep1ffic . Thus,
salt concentration can be used as a knob to either maximize or
remove the electrostatic interaction through the exponential part,
even in the case of large σ s and σ t. While high salt concentration
decreases λD to low values and suppress electrostatics until very
short z (sharp kink in FZ), low salt concentration increases λD and
facilitates the detection of electrostatics (soft kink in FZ). For the
monovalent NaCl salt concentration of 2 mM,
ffiffiffiffiffiffiffiffiffi ¼ 6:8 nm [63]. If we use HOPG as the supporting
λD ¼ p0:304
0:002
surface for protein cages (Fig. 9a), the neutral character of this
substrate would suppress Fel. However, electrostatics will be detect-
able when the protein shell is indented, if there exists any density of
charge on it. Figure 9b shows nanoindentations performed on the
HOPG substrate (dark) and the virus (red) of Fig. 9a. While FZs on
HOPG show a sharp kink just before contact due to FvdW, indenta-
tion data on virus show a soft landing of the cantilever due to
electrostatic repulsion Fel. The charge of a particle can be estimated
by fitting nanoindentation curves to two-sphere DLVO models
[64]. The charge of hAdV and Minute Virus of Mice (MVM) can
also be derived from their structure [65] and compared with the
experiments (Fig. 9c), reaching a reasonable agreement. Interest-
ingly, the presence of dsDNA affects the charge density of viruses
(Fig. 9d), although the underlying mechanism of this influence
remains unknown [64].
274 Álvaro Ortega-Esteban et al.

Fig. 9 Electrostatics. (a) AFM topography of a phi29 bacteriophage on HOPG. (b) FZs performed along the
labels of a): HOPG (dark) and particle (red). (c) Comparison between the estimated charge from virus structure
[65] and the measured values. (d) Influence of DNA on the charge of virus particles (Adapted from [64])

8 Conclusions

AFM enables new possibilities for studying individual virus parti-

cles, complementing classical EM and X-ray diffraction studies.
First, AFM may routinely work with individual viral particles in
buffer conditions, which are closer to the in vivo medium than
the typical structural biology techniques. Although the AFM imag-
ing resolution is below virus structures obtained with structural
biology techniques, AFM enables the identification of virus ele-
ments which are not symmetrically ordered. Second, structural
virology studies are typically focused on establishing structure-
function relationships. Nevertheless, AFM can study some physical
properties of viral particles in real time which can complement the
above binomial to become a trinomial: structure-function-prop-
erty. On the other hand, AFM offers the possibility of manipulating
 AFM of Protein Shells 275

individual virus capsids by inducing the shell disruption with

mechanical stress. These experiments contribute to understand
the interbond interactions, which throw light on virus assembly/
disassembly.

Acknowledgements

We acknowledge our collaborators and projects FIS2017-89549-

R, Fundación BBVA and “Marı́a de Maeztu” Program for Units of
Excellence in R&D (MDM-2014-0377).

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