Structure of the KvAP voltage-dependent K?channel
and its dependence on the lipid membrane
Seok-Yong Lee, Alice Lee, Jiayun Chen, and Roderick MacKinnon*
Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, 1230 York Avenue,
New York, NY 10021
Contributed by Roderick MacKinnon, September 7, 2005
Voltage-dependent ion channels gate open in response to changes
in cell membrane voltage. This form of gating permits the propa-
gation of action potentials. We present two structures of the
voltage-dependent K?channel KvAP, in complex with monoclonal
studied KvAP with disulfide cross-bridges in lipid membranes.
and EPR data on KvAP we reach the following conclusions: (i) KvAP
is similar in structure to Kv1.2 with a very modest difference in the
orientation of its voltage sensor; (ii) mAb fragments are not the
source of non-native conformations of KvAP in crystal structures;
(iii) because KvAP contains separate loosely adherent domains, a
lipid membrane is required to maintain their correct relative
orientations, and (iv) the model of KvAP is consistent with the
proposal of voltage sensing through the movement of an arginine-
containing helix–turn–helix element at the protein–lipid interface.
membrane protein ? protein-lipid interface ? voltage-gated ion channel ?
its value (1). These channels contain a centrally located pore
surrounded by four voltage sensors. Voltage-dependent K?(Kv)
channels are tetramers with four identical subunits, each with six
transmembrane segments (S1–S6): S5 and S6 form the central
pore at the interface between the subunits, and S1–S4 form the
voltage sensors (2, 3). The voltage sensors have four to seven
positively charged amino acids (usually arginine) on S4, known
as gating charges, and fewer negatively charged amino acids
(aspartate or glutamate) distributed on S1, S2, and S3. The
voltage sensors undergo a conformational change when the pore
gates open, coupling movement of the S4 gating charges within
the membrane electric field to channel open probability.
The first structure of a Kv channel, termed KvAP, from the
archeabacterium Aeropyrum Pernix (4), was determined by crys-
tallizing the channel as a complex with a monoclonal Fab
fragment attached to its voltage sensors [Protein Data Bank
(PDB) ID code 1ORQ] (5). In that structure the voltage sensors
are in a non-native conformation, displaced toward the intra-
cellular side of the transmembrane pore. Together with a second
structure of the voltage sensor alone, termed the isolated voltage
sensor (PDB ID code 1ORS), the following ideas about voltage-
dependent gating were proposed (5, 6): that the voltage sensor
is a very mobile structure, presumably because it must carry
charged amino acids through the membrane electric field; that
the charge bearing S4 forms a helix–turn–helix with the C-
terminal half of S3 (S3b), and that the helix–turn–helix moves at
the protein–lipid interface a large distance. Experiments using
avidin capture of biotin linked to the voltage sensor indicated
that four of the S4 arginine amino acids translate 15–20 Å across
the membrane. These structural and functional studies formed
the basis for a conceptual model in which the helix–turn–helix
‘‘paddle’’ element shifts its position at the protein–lipid inter-
face, opening the pore while moving its charged amino acids (6).
oltage-dependent ion channels ‘‘sense’’ voltage differences
across the cell membrane and open or close in response to
Because of the non-native conformation of the voltage sensors
in the crystal, the KvAP structure left two compelling questions
unanswered. The first, which is important for understanding the
mechanism of voltage-dependent gating, is what is the native
membrane conformation of KvAP? The second question, re-
lated to the mechanism of gating but also relevant to the more
general subject of membrane protein structure, is why does
KvAP not maintain a native conformation in crystals? Much
speculation surrounded this second question, including the
wonder whether antibody fragments can ‘‘distort’’ membrane
This study attempts to address both questions. We have
determined two additional structures of KvAP, bound to mono-
clonal Fv fragments and in the absence of antibody fragments
altogether, and we have carried out cross-link studies with KvAP
in lipid membranes. Analysis of these data in the context of the
crystal structure of a eukaryotic relative of KvAP, known as
Kv1.2 (7, 8), and in the context of published EPR data on KvAP
(9), leads to a constrained model for a native structure of KvAP
in lipid membranes. The results also lead to an interesting
general conclusion about the structure of Kv channels: because
they are comprised of separate loosely adherent domains (pore
and voltage sensors) embedded in the membrane, an intact lipid
membrane is required to maintain a native orientation of
domains with respect to each other. We anticipate that other
complex membrane proteins exhibiting this property will be
identified in the future, and discernment of their structures will
require a combination of structural and biochemical techniques,
similar to the approaches used here.
Structure Determination. The KvAP channel was expressed and
purified as described (5) with modifications (Supporting Text,
which is published as supporting information on the PNAS web
site). Monoclonal Fv fragments were produced by expression in
Escherichia coli and purified by using Co2?affinity and gel
filtration chromatography. A first crystal form was grown by
vapor diffusion in the absence antibody fragments, and maps
were calculated by using density modified molecular replace-
by vapor diffusion in the presence of Fv fragments, phases were
determined by molecular replacement and heavy atom deriviti-
zation by using Tl?(10), and an atomic model was built and
refined (Table 1, which is published as supporting information
on the PNAS web site) (11, 12).
Cross-Bridge Studies. Biochemical cross-bridge studies on KvAP
were carried out after introducing cysteine residues at specific
Freely available online through the PNAS open access option.
Abbreviations: KV, voltage-dependent K?; PDB, Protein Data Bank.
Bank, www.pdb.org (PDB ID code 2A0L).
*To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
October 25, 2005 ?
vol. 102 ?
no. 43 ?
locations in the pore and voltage sensor by using QuikChange
(Stratagene). Inside-out membrane vesicles were prepared as
described with slight modification (13). Air oxidation (no cata-
lysts) was allowed to occur in membranes overnight at room
temperature. N-ethyl-maleimide (20 mM) was added to the
vesicles before SDS solubilization to block remaining free cys-
teines. Western blot was carried out by using an antibody raised
against the KvAP channel. Further details are provided in
Crystal Structures With and Without Antibody Fragments. The struc-
ture determinations were carried out by using a truncated
version of KvAP (36 C-terminal amino acids deleted). The
deleted amino acids follow the sixth (last) membrane-spanning
?-helix and were disordered in the original structure of KvAP.
The function of the truncated channel in lipid membranes is very
similar to the full-length KvAP channel.
Two additional crystal forms were grown by using the deter-
gent-like lipid 1,2-diheptanoyl-sn-glycero-3-phosphocholine.
The most important difference between these crystals is that one
was grown in the absence of antibody fragments and the other
as a complex with monoclonal Fv fragments attached to the
voltage sensors. Triclinic crystals of KvAP without antibody
fragments diffracted to a resolution of 8 Å. Molecular replace-
ment using the KvAP pore without voltage sensors (from PDB
ID code 1ORQ) showed two channels in the unit cell (10). An
electron density map shows clear helical segments for the pore
and the voltage sensors, which were absent in the model phases
(Fig. 1a). The helical organization of KvAP is discernable at this
resolution. In contrast to the initial structure of KvAP (PDB ID
code 1ORQ), the voltage sensors are located entirely within the
‘‘plane of the lipid membrane.’’ The crystal lattice is supported
by contacts between the helix–turn–helix voltage sensor paddle
elements, which make contacts with each other from adjacent
channels (Fig. 1b).
Tetragonal crystals of KvAP in complex with Fv fragments
diffracted X-rays to a resolution of 3.9 Å. Molecular-
replacement phases using the KvAP pore were combined with
heavy atom phases from Tl?-soaked crystals (10). After solvent
flattening and averaging, a good-quality electron density map
(Fig. 1c) allowed building of a model and refinement to Rfree0.39
(10–12). This crystal lattice is mediated by contacts between Fv
fragments (Fig. 1d).
The ?-carbon trace of KvAP in the 8-Å electron density map
(Fig. 1a) is the KvAP–Fv complex structure after removal of the
Fv fragments. It is evident that the channel structures in the two
crystal forms are very similar even though one has Fv fragments
bound to its voltage sensors and the other does not. The Fv
fragments have not had a significant influence on the confor-
mation of the voltage sensors. Because the KvAP–Fv complex
structure is better defined it will be the focus of further
Comparison with Previous KvAP Structures.Thestructureofasingle
subunit from the KvAP tetramer (Fig. 2a) is shown from the side
with the extracellular surface above. The essential features of
this structure include voltage sensors (helices S1–S4) surround-
ing the centrally located pore, S1 and S2 helices wrapped around
the pore, and helix–turn–helix S3b–S4 voltage sensor paddles
positioned adjacent to the S2 helices. The major difference
between this structure and the original KvAP crystal structure
(Fig. 2b; PDB ID code 1ORQ) is the position of the paddle,
which in this structure is drawn near to S2 at the level of the
membrane rather than extended toward the cytoplasm.
In this structure the position of the paddle with respect to S2
is very similar to that observed in the crystal structure of the
isolated voltage sensor, in which Arg-133 on S4 forms a salt
bridge with Asp-62 on S2 (PDB ID code 1ORS) (Fig. 2c). The
same salt bridge is present in the full-length channel structure.
crystals of the original KvAP structure (in which the paddle is
pulled away from S2 and the salt bridge is broken) were grown
at pH 5.0 (5), crystals of the isolated voltage sensor were grown
at pH 7.5 (5), and crystals of the KvAP-Fv and KvAP (8 Å)
crystals were grown at pH 8.5 and 9.0, respectively. Thus,
conditions that favor salt-bridge formation (higher pH) support
a conformation in which the paddle remains near S2, within the
plane of the membrane. This conformation of the paddle with
respect to S2 and salt-bridge formation involving Arg-133 is now
observed in more than one crystal structure of KvAP. Shaker K?
channels do not function when the corresponding arginine
residue (position 377) is mutated (2). For these reasons we
suspect that the observed salt bridge and position of the voltage
sensor paddle with respect to the S2 helix is functionally relevant.
The S1 and S2 helices in the structures have the same
horizontal disposition as seen previously, although in mem-
branes we know they must have a more vertical orientation (9,
23) (Fig. 2 a and b). In this regard the crystal structures in this
study, like the original structure, do not represent a truly native
conformation of the voltage sensor. The horizontal positioning
of S1 and S2 cannot be attributed to the antibody fragments
bound to the voltage sensor because we observe the same
conformation in three structures: with Fab fragments (5), with
Fv fragments, and without antibody fragments. If not antibody
fragments, what accounts for the horizontal positioning of these
fragments. (a) Four-fold averaged electron density map (1.0 ?) of KvAP at 8 Å
Two KvAP pores were used to calculate the map. The ?-carbon traces of the
complex structure onto the KvAP pore molecular replacement solutions. (b)
Contacts in the KvAP crystal. A layer of channel molecules is formed by lateral
and end-to-end packing between layers. Two channels (red), surrounded by
black lines, define the unit cell. (c) Native sharpened 2-fold averaged electron
density map (1.0 ?) of the KvAP–33H1 Fv complex at 3.9 Å calculated with Fo
Fourier coefficients. Combined phases (single isomorphous replacement with
anomalous scattering and partial molecular replacement phases) were used
for map calculation. The ?-carbon traces of the KvAP–Fv structure were
down the crystallographic 4-fold axis. Channels are colored blue, and Fv
fragments are colored green. Four channels and 16 Fvs, surrounded by black
lines, comprise a unit cell.
KvAP structures are in similar conformations with and without Fv
www.pnas.org?cgi?doi?10.1073?pnas.0507651102 Lee et al.
Role of the Lipid Bilayer in Voltage Sensor Orientation. Several
features of the crystal structures in this study provide a likely
explanation for the horizontal orientation of the voltage sensor
in detergent micelles. The rather elaborate turn connecting S2 to
S3 contains five tyrosine amino acids that project out from its
perimeter (Fig. 3a). In the crystal this turn is located at a level
corresponding to the middle of the membrane (Fig. 2a), which
is unexpected because tyrosine side chains are energetically
favored to reside at the interface between the hydrophobic and
The detergent-like lipid 1,2-diheptanoyl-sn-glycero-3-phospho-
choline used to crystallize the channel does not form planar lipid
membranes, only micelles, so a planar membrane constraint is
absent in the crystal. In the context of a lipid membrane with
planar boundaries we should expect the tyrosine residues to be
driven toward the interface layer. A rigid body rotation of a
voltage sensor domain (or of helices S2–S4) around its connec-
tion to S5, as shown (Fig. 3b), would place the tyrosine residues
at the interface. The same rotation would also bring the S1–S2
loop (which has near it several tyrosine residues) to the extra-
cellular lipid–water interface. From these energetic consider-
ations we suggest that a planar lipid membrane constrains the
voltage sensor domains with respect to the pore, and when the
channel is removed from the membrane, the unconstrained
domains can easily adopt a non-native orientation. This behavior
is interesting because it would be expected to occur only if the
domains are loosely attached to the pore as fairly self-contained
Several lines of evidence support the idea that the voltage
sensors do indeed function as nearly self-contained structural
(in the absence of a pore) enabled us to determine its structure,
and upon expression the domain is targeted to and stable in the
cell membrane (5). Second, voltage dependence is transferable
by splicing a voltage sensor onto a nonvoltage-dependent K?
channel (17). And third, a voltage-dependent phosphatase with
an S1–S4 sequence attached to a soluble enzyme shows that the
voltage sensor can operate on a target other than an ion
Defining the Position of the KvAP Voltage Sensor by Analogy to Kv1.2.
The structure of a related Kv channel, Kv1.2 from the rat, helps
to define the native position of the KvAP voltage sensor with
respect to the pore (Fig. 4a) (PDB ID code 2A79) (8). Potential
distortions of the Kv1.2 voltage sensor upon extraction from the
membrane are restricted by the connection of the S1 helix to a
cytoplasmic domain (not shown in Fig. 4a), which does not exist
in KvAP. The KvAP voltage sensor (Fig. 2a) can be adjusted to
have the appearance of the sensor in Kv1.2 by rotating the sensor
vertically (as was done in Fig. 3b), straightening the S4–S5 linker
helix, and folding the S1 helix back onto the body of the sensor,
similar to its position in the KvAP isolated voltage sensor crystal
structure (PDB ID code 1ORS) (Fig. 4b). In the resulting KvAP
model, the S4–S5 linker is like that in Kv1.2: it forms an
amphipathic helix with a hydrophobic surface (to face the
membrane) and a hydrophilic surface (to face the cytoplasm)
(Fig. 5a). Furthermore, in KvAP the sharp turn connecting the
S4 transmembrane helix to the S4–S5 linker interfacial helix
occurs at the position of a glycine amino, which is frequently
found at such turns. The straightforward transformation from
the crystal structures of KvAP to the model in Fig. 4b, with
preservation of chemical features that correspond well to those
are shown as ball-and-stick representation. (b) Possible transformation of the
voltage sensor upon detergent extraction. (Left) The structure of KvAP in a
detergent micelle. S2, S3, and S4 helices are colored blue, and the turn
connecting S2 to S3 helices is colored green. (Right) The putative conforma-
tion of KvAP in the membrane. Asterisk marks the pivot point of proposed
voltage sensor rotation.
The structure of the S2 to S3 turn. (a) The turn connecting the S2 to
KvAP structures. (a) Stereo view of a single KvAP subunit from the side with
a single KvAP subunit of the KvAP–6E1 Fab complex (PDB ID code 1ORQ). (c)
Comparison with the isolated voltage sensor structure. Stereo view of a
superposition of the isolated voltage sensor (gold, PDB ID code 1ORS) and the
KvAP–Fv complex (blue). The S2 helix (Leu-55 to Tyr-75) was used for super-
position, and the S1 helix is not shown. Residues Asp-62, Asp-72, Arg-76,
Glu-93, and Arg-133, which are important for channel function, are shown in
Structure of the KvAP–33HI complex and comparison with previous
Lee et al.
October 25, 2005 ?
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in Kv1.2, provides good evidence that the native structure of
KvAP is similar to Kv1.2.
Biochemical Information on KvAP in Lipid Membranes. Perozo and
colleagues (9) have used EPR spectroscopy to measure lipid
exposure of protein surfaces of the KvAP voltage sensor. These
data agree well with the position of the voltage sensor in Kv1.2
(8) (Fig. 4a) with one small discrepancy. In the Kv1.2 crystal
structure, one edge of the S4 helix leans against the S5 helix of
a neighboring subunit; however, the corresponding amino acids
Kv1.2. (a) A subunit of the Kv1.2 (PDB ID code 2A79)
viewed from the side. The voltage sensor region (S1–
S4) is colored blue, and the pore region (S5–S6) is
colored gray. A queue of K?ions (green spheres) is
shown as a reference point for comparison with the
KvAP model. (b) The KvAP model. The model was
constructed by tilting S2–S4 helices of the KvAP struc-
ture (PDB ID code 2A0L), adjusting the S4–S5 linker to
2A79), folding the S1 helix similar to its position in the
isolated voltage sensor (PDB ID code 1ORS), and repo-
sitioning the sensor slightly to account for EPR O2
(lipid) accessibility (9).
A model of KvAP based on the structure of
chains of the S4–S5 linker shown as a ball-and-stick representation indicate the amphipathic nature of this linker region. (b and c) The EPR O2(lipid) accessibility
data (9) mapped onto the voltage sensor structure. O2accessibility ranges from white (low accessibility) to red (high accessibility). Numbers in c indicate amino
acid position, circles show positions where data are not available. (d) Western blot analysis of cross-linking. Membrane vesicles containing single or double Cys
faster than the other cross-linked tetramers.
Mapping EPR O2(lipid) accessibility data onto the voltage sensor region of the KvAP. (a) The S4–S5 linker (colored blue) of the KvAP model. The side
www.pnas.org?cgi?doi?10.1073?pnas.0507651102Lee et al.
in KvAP exhibit high lipid exposure according to the EPR data
(Fig. 5 b and c, residues 124 and 127). In addition, a face of S1
in Kv1.2 that would be expected to exhibit high lipid exposure
(because it is on the lipid-facing surface) appears in KvAP to be
in a protein environment (i.e., neither lipid nor water) according
to the EPR data (Fig. 5 b and c, residues 31, 35, and 39). These
differences between expected lipid exposure on the basis of the
Kv1.2 crystal structure and observed lipid exposure of corre-
sponding amino acids in KvAP suggest that the voltage sensor in
KvAP might be rotated slightly with respect to the pore,
compared with the sensor in Kv1.2. The rotation of the voltage
modest and in fact has already been made in the model shown
in Fig. 4b, relative to the Kv1.2 voltage sensor in Fig. 4a. That
small variations in the orientation of the voltage sensors might
occur between KvAP and Kv1.2 is not surprising, especially
because the constraint imposed by the linker to the T1 domain
We carried out disulfide cross-bridge studies to further
examine the relationship between KvAP and Kv1.2. Several
studies have shown that a cysteine residue introduced at the
position of the first arginine residue on S4 (the extracellular-
most arginine, corresponding to position 117 in KvAP) in the
Shaker K?channel (which is very similar to Kv1.2) readily
forms a cross-bridge with a cysteine introduced near the
extracellular end of S5 of a neighboring subunit (19). We
observe a qualitatively similar result with KvAP channels in
membranes (Fig. 5d). In KvAP, however, we do not observe
the same quantitative degree of complete covalent tetramer
formation that is observed in the Shaker K?channel (19). The
data on KvAP are thus consistent with the voltage sensor being
in a similar location as in Shaker (or Kv1.2), but the lower
efficiency of tetramer formation is consistent with the possi-
bility that S4 may be slightly further away from S5, as the EPR
We note additional aspects of the disulfide cross-bridge stud-
ies on KvAP channels that parallel previous data on Shaker K?
channels. Cross-bridge formation between cysteine residues on
the voltage sensor paddle and the extracellular side of S5 do not
depend highly on the precise location of the cysteines (19–22),
and a single cysteine residue near the extracellular ‘‘tip’’ of the
voltage sensor paddle can result in the formation of covalent
subunit dimers, presumably through linkage of voltage sensor
paddles from adjacent subunits (20). The nonspecificity of
cross-bridge formation and covalent subunit dimerization me-
diated by single cysteine residues on the voltage sensor paddle
suggests that the paddle is a highly mobile unit (Fig. 5d).
Two additional crystal structures of KvAP show the voltage
sensors within the plane of the lipid membrane, but the domains
still deviate from their expected vertical orientation relative to
the pore. Antibody fragments per se are not the cause of this
deviation because it is observed in the presence of Fab fragments
and Fv fragments and in the absence of antibody fragments.
Therefore, it is incorrect to conclude that antibody fragments
should be avoided in the crystallization of membrane proteins.
One important conclusion of this study, and the answer to the
second question raised in the Introduction, is that KvAP appears
to require an intact lipid membrane to maintain a native
conformation, because the voltage sensors do not adhere tightly
to the pore. The sensors, like the pore, have their own intrinsic
structural and chemical properties to match the membrane’s
hydrophobic core, interface, and water layers, ensuring that they
first examples of membrane proteins with separate, weakly
attached membrane-spanning domains, but other important
membrane proteins with this property will likely be identified
and studied in the future.
For proteins like KvAP the detergent micelle may be an
imperfect mimic of the lipid bilayer, but detergent-mediated
crystallization is still a valuable approach for understanding such
proteins. A non-native protein structure simply means that
additional information is required to help interpret what we see.
In the present analysis we have combined crystal structures of
the KvAP channel with biochemical cross-bridge studies in
of the voltage sensor relative to the pore.
A model of the KvAP tetramer in the open conformation. The top-down view (a) and the side view (b) of the proposed model of the KvAP tetramer
Lee et al.
October 25, 2005 ?
vol. 102 ?
no. 43 ?
a related Kv channel structure to arrive at a plausible answer to
the first question raised in the Introduction: what is the native
structure of KvAP in lipid membranes? The model is shown in
Fig. 6. Coordinates for the model shown in Fig. 6 are available
on request from R.M.
This proposed KvAP structure is quite similar to the Kv1.2
channel structure, which was used to help guide its construction
(8). The pore conformation corresponds to that observed in two
KvAP crystal structures (PDB ID codes 1ORQ and 2A0L) with
the S6 inner helices adjusted slightly to accommodate their
interaction with the S4–S5 linker helices. The S6 inner helices
near the intracellular pore entryway have a wide diameter (?12
Å), suggesting that the pore is open. The conformation of the
voltage sensors is very similar to the sensor in the crystal
structure of KvAP (PDB ID code 2A0L) but with S1 moved near
to its position in the isolated voltage sensor structure (PDB ID
code 1ORS) (5). The voltage sensor domains in KvAP, com-
pared with those in Kv1.2, are rotated slightly about an axis
perpendicular to the membrane plane relative to the pore in such
a way that S1 is more buried in protein and S4 is more exposed
to lipid to account for lipid exposed surfaces mapped by EPR
analysis (9). The conformation of the voltage sensor paddle (the
helix–turn–helix formed by S3b and S4) is near to an open
conformation because the four conserved arginine residues on
S4 are near the extracellular surface.
The turn connecting S2 to S3, which is not resolved in the
structure of Kv1.2, is defined by several crystal structures of
KvAP. On the basis of amino acid sequence analysis we think
that this turn will be conserved in voltage-dependent K?and
Na?channels. In particular, there is a salt-bridge pair that is
always conserved, with a nearly fixed number of amino acids
between them (see Fig. 7, which is published as supporting
information on the PNAS web site). In KvAP, and probably
other voltage-dependent channels, we propose that one of the
functions of this turn is to serve as a membrane interface anchor.
The various crystal structures of KvAP suggest that this turn also
serves a second function, to allow the voltage sensor paddle to
undergo large movements. We base this statement on the
observation that the turn is different in its conformation from
one crystal structure to the next, indicating that it is intrinsically
flexible (PDB ID codes 1ORS, 1ORQ, and 2A0L).
The model of KvAP shown in Fig. 6, which is well constrained
by crystallographic data, biochemical data, and the structure of
Kv1.2, is in good agreement with the original conceptual model
for KvAP gating in which an arginine-containing helix–turn–
helix paddle element was proposed to move at the protein–lipid
interface to control gating (6). Recent experiments on the
accessibility of avidin to tethered biotin of various lengths
attached throughout the KvAP protein are in excellent agree-
ment with this model (23).
We thank the staffs for beam lines ALS 8.2.1 (Advanced Light Source,
Lawrence Berkeley National Laboratory, Berkeley, CA), CHESS A1
(Cornell High Energy Synchrotron Source, Cornell University, Ithaca,
NY), and NSLS X-25 (National Synchrotron Light Source, Brookhaven
F. Valiyaveetil, and R. Jain for advice. This work was supported by
Coffin Childs Memorial Fund fellow, and R.M. is an Investigator in
Howard Hughes Medical Institute.
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