Structure of the human glucagon class B
Jeremiah S. Joseph1, Wei Liu1, Jesper Lau5, Vadim Cherezov1, Vsevolod Katritch1, Ming-Wei Wang2& Raymond C. Stevens1
helical domain of human GCGR at 3.4A˚resolution, complemented by extensive site-specific mutagenesis, and a hybrid
shared seven transmembrane fold, the GCGR transmembrane domain deviates from class A G-protein-coupled receptors
turns above the plane of the membrane. The stalk positions the extracellular domain ( 12kilodaltons) relative to the
membrane to form the glucagon-binding site that captures the peptide and facilitates the insertion of glucagon’s amino
terminus into the seven transmembrane domain.
GCGR is activated by the 29 amino acid hormonal peptide glucagon
During fasting, the pancreas dispatches glucagon to activate GCGR in
class B GPCRs, many of these receptors presumably share a seven
mechanisms1. Although the structure–function understanding of the
classA family ofGPCRs has been greatly advancedduring the lastfew
lack of a 7TM domain structure for these receptors.
Secretin-like GPCRs contain a globular N-terminal extracellular
domain (ECD) defined by three conserved disulphide bonds4,5and a
7TM domain. They are activated by hormonal peptides that bind to
of peptide hormones’ carboxy termini have been revealed for several
binding have been proposed based on site-directed mutagenesis9–11,
photo-crosslinking12–14, and structure-based virtual screening studies15,
homology between class A and class B GPCRs.
Crystal structure of GCGR 7TM domain
The 7TM domain of human GCGR was fused to the thermally stabi-
lized E. coli apocytochrome b562RIL (ref. 16) (referred to as BRIL) at
residue 123, and the C terminus of GCGR was truncated at residue
432 (Supplementary Fig. 2). This crystallized GCGR construct with
BRIL containing a truncated ECD (DECD) and C terminus (DC)
that the conformation of the 7TM domain of BRIL–GCGR(DECD/
DC) is similar to wild-type GCGR. The structure of the BRIL–
GCGR(DECD/DC) was determined at 3.4A˚resolution (Methods
and Supplementary Table 2). Although GCGR was crystallized in
the presence of NNC0640, convincing electron density for NNC0640
the BRIL fusion protein folded on top of the receptor and mediating
most of the crystal contacts (Supplementary Fig. 4).
Despite the lack of protein sequence conservation, comparison of
the GCGR 7TM structure with 15 known class A GPCR structures
in the 7TM bundles are conserved between the two classes (Fig. 1b,
Supplementary Fig. 5). The 7TM helices of GCGR superimpose with
structural alignment of GCGR with rhodopsin shows an approximate
GPCR classes, but also reveals a number of gaps in transmembrane
regions reflecting substantial structural deviations in transmembrane
helices (Supplementary Fig. 6). The spatial correspondence between
7TM residues makes it possible to project the widely used class A
Ballesteros-Weinstein numbering scheme17(used hereafter for class
A as BW number in parentheses) for comparisons between GPCR
classes (Supplementary Table 3). Analysis of sequence and structural
features within class B GPCRs, however, is defined by the Wootten
after for class B receptors as superscript, Supplementary Table 3).
Class B versus A GPCRs
that are distinct from known class A GPCRs. The N-terminal end of
helix I in GCGR is longer than any known class A GPCR structures
and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 189 Guo Shou Jing Road, Shanghai, 201203, China.3Division of Medicinal
Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), VU University of Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.4The Joint
Nordisk Park, 2760 Malov, Denmark.
4 4 4 | N A T U R E | V O L 4 9 9 | 2 5 J U LY 2 0 1 3
Macmillan Publishers Limited. All rights reserved
the extracellular (EC) membrane boundary from Lys136 to Gly125
in glucagon binding and helps to define the orientation of the ECD
is 16 residues long, as compared with 4–6 residues in most class A
GPCRs. Although the ECL1 residues 201–215 are not resolved in the
indicate that these residues are involved in interactions with peptide
ligands. The distance between the EC tips of 7TM helices II and VI
tioning of the EC tips of these 7TM helices creates a wider and deeper
cavity in the ligand-binding pocket of GCGR, which is larger than in
any class A receptor structures (Fig. 2, Supplementary Table 4).
At the intracellular (IC) side, the distances between the helical tips
of GCGR are within the same range as those in class A structures,
except for an extensive inward shift of the IC tip of helix VII (Sup-
plementary Fig. 5). Although the inward shift in the IC part of helix
role the IC region of helix VII plays in GCGR. The receptor lacks a
proline kink in helix VII, which is a part of the conserved NP (BW
7.50) xxY motif in class A GPCRs24; instead, helix VII of GCGR has a
motif that is fully conserved in secretin-likeclass B receptors (Fig. 3a).
This Gly3937.50induced bend is stabilized by hydrophobic interac-
tions with Phe1842.57of helix II in GCGR (Fig. 3a).
The GCGR structure also includes an IC helix VIII comprising 20
residues at the C-terminal end of the receptor that tilts approximately
25u away fromthe membraneas comparedwithits consensus position
in class A (Fig. 1). This tilt is probably a result of crystal packing inter-
actions (Supplementary Fig. 4), but it should be noted that Glu406 in
two interhelical salt bridges with conserved residues Arg1732.46and
in the region, conformational modelling with helix VIII parallel to the
membrane suggests that the Glu406 salt bridges are preserved in this
Figure 2 | Comparison of the ligand-binding pocket of GCGR with class A
GPCRs. The binding cavity of GCGR is compared with the binding cavities
of human chemokine receptor CXCR4 (PDB: 3ODU), human k-opioid
receptor (k-OR) (PDB: 4DJH), rat neurotensin receptor (NTSR1) (PDB:
4GRV), human b2-adrenergic receptor (b2AR) (PDB: 2RH1) and bovine
rhodopsin (Rho) (PDB: 1U19) (Supplementary Table 4). The approximate
position of the EC membrane boundary is shown as a red line and bound
ligands as magenta carbon atoms.
III III IV IV
VIII VIII VIIIVIII
VIII VIII VIII
Figure 1 | Structure of the 7TM domain of human GCGR and comparison
to class A GPCR structures. a, Cartoon depiction of the 7TM domain
structure of GCGR. The two views are rotated 180u relative to each other. The
disulphide bond between helix III and extracellular loop 2 (ECL2) is shown as
yellow sticks. b, Side view of structural superimposition of 7TM domains of
GCGR (blue) and class A GPCRs (grey). Structures of class A GPCRs used
(PDB): 1U19, 2RH1, 2YCW, 3RZE, 3PBL, 3UON, 4DAJ, 3EML, 3V2W,
3ODU, 4DJH, 4EA3, 4DKL, 4EJ4 and 3VW7. Extracellular (EC) and
intracellular (IC) membrane boundaries (predicted by OMP server44) are
shown as brown and cyan ovals (a) or dotted lines (b), respectively.
2 5 J U LY 2 0 1 3 | V O L 4 9 9 | N A T U R E | 4 4 5
Macmillan Publishers Limited. All rights reserved
B receptors because there is no strong conservation among these resi-
dues in class A.
The GCGR 7TM structure also reveals several structural features
that are conserved between class A and B receptors. One such feature
is a disulphide bond between Cys294 in ECL2 and Cys2243.29(BW
7TM fold (Fig. 3c). Another conserved feature of a common GPCR
fold25involves similar regions of contacts between helices I–II, I–VII,
III–IV and III–VI in class A and B GPCRs. The two GPCR classes,
tions (Fig. 3, Supplementary Fig. 6). In class B GPCRs, the helix I–II
interaction is stabilized byconserved hydrophobicresidues Leu1561.54
and Phe1842.57, class A GPCRs contain conserved polar residues Asn
(BW 1.50) and Asp (BW 2.50) in this region25. At the helix I–VII inter-
(Fig. 3a). Mutation of homologous glucagon-like peptide-1 receptor
(GLP1R) residues Ser1551.50and Ser3927.47alters receptor signalling18.
At the GCGR helix III–IV interface (Fig. 3d), the conserved residue
Trp residue in helix IV (BW 4.50) interacts with the residue at BW
24, 25). The helix III–VI interface (Fig. 3e) in secretin-like class B
GPCRs contains conserved hydrophobic residues Tyr2393.44(or
Phe3.44) and Leu3586.49(or Phe6.49) which make similar hydrophobic
interactions as structurally aligned Ile/Val/Leu (BW 3.40) and Phe
(BW 6.44) residues present in most class A GPCRs25(Supplementary
Fig. 6). This interface is further stabilized in class B GPCRs by close
contact between the conserved Tyr2393.44and Gly3596.50. Another
class B GPCR specific interhelical hydrogen bond is formed between
the conserved Asn3185.50and the backbone of Leu2423.47at the helix
III–V interface (Fig. 3e).
Recognition between GCGR and glucagon
To better understand GCGR–glucagon interactions, we performed a
comprehensive mutagenesis and glucagon-binding study of GCGR at
90 different residue positions (Fig. 4, Supplementary Table 5). A total
of 129 mutants were tested, and of these, 110 covering 85 different
positions had expression levels greater than 30% of wild-type GCGR.
Of them, 41 mutations covering 28 different positions in the GCGR
7TM domain had more than fourfold reduction in glucagon binding
(IC50values) relative to wild-type GCGR. The results of these GCGR
in glucagon binding face the main cavity in the 7TM core, and form a
binding site that covers parts of ECL1, ECL2 and ECL3 and helices I,
II, III, V, VI and VII, and extends deep into the 7TM cavity.
To investigate the recognition between glucagon and GCGR, we
built a glucagon-bound GCGR structure model, based on the GCGR
conformation of pituitary adenylate cyclase activating polypeptide
(PACAP; PDB: 1GEA)26(Fig. 5a). The model further included several
The predicted binding mode of glucagon to the ECD of GCGR
(Fig. 5b) is in line with our results (Fig. 4, Supplementary Table 5)
and previously reported mutation studies on GCGR6,27and GLP1R8.
Figure 5b shows how GCGR residues Asp63, Tyr65 and Lys98 func-
tion in stabilizing the ECD as observed in the GCGR ECD crystal
structure6and supported by mutagenesis studies6,27(Fig. 4, Sup-
plementary Table 5). The Trp36 side chain is an important hydro-
phobic interaction site for the C-terminal region of glucagon, similar
to Trp39 in the GLP1–GLP1R-ECD crystal structure8. The stalk,
observed in helix I of the GCGR 7TM crystal structure, links the
ECD and 7TM domain in the model (Figs 5a, b). The a-helical con-
formation of the stalk is supported by intrahelical interactions in the
crystal structure (Glu133–Lys136) and model (Glu127–Gln131 and
Glu129–Lys132), and is likely to be further stabilized by interactions
with the extended ECL1 and the a-helical portion of glucagon. The
potential function of the a-helical stalk in glucagon binding is sup-
ported by a reduced glucagon affinity to the Ala135Pro mutant
Helix III – V – VI Helix III – V – VI
Helix III – V – VI
Disulfide bondDisulfide bond
Helix VIII ionic networkHelix VIII ionic network
Helix VIII ionic network
E406 E406 E406
Helix I – II – VIIHelix I – II – VII
Helix I – II – VII
I IIII II IIVII VII VII
Helix III – IVHelix III – IV
Helix III – IV
Figure 3 | Structural features of
class B GPCRs. Comparison of
GCGR and class A GPCR crystal
structures indicates distinct and
conserved features. a, d, e, The
homologous GCGR residues
involved in helix I–II, III–IV, and
III–VI interface interactions as
discussed for class A receptors by
Venkatakrishnan et al.25, and class B
GPCR specific residues that mediate
helix I–VII, II–VII, and III–V
interface interactions. b, GCGR
residues Glu406 of helix VIII,
Arg1732.46, and Arg3466.37form a
class B receptor specific ionic
network. Arg3466.37(grey) has a
weak electron density. c, The
disulphide bond between Cys2243.29
and Cys294 of ECL2 in the GCGR
structure is a conserved feature
between classes A and B receptors.
Hydrogen bond interactions and salt
lines. Electron density maps for
residues in this figure are shown in
Supplementary Fig. 10. Comparison
of class A Ballesteros–Weinstein and
provided in Supplementary Table 3.
4 4 6 | N A T U R E | V O L 4 9 9 | 2 5 J U LY 2 0 1 3
Macmillan Publishers Limited. All rights reserved
the binding site for the previously proposed middle hinge region of
glucagon28. An a-helical conformation in this region of GLP1R in
linking data between receptor and peptide residues12. In the GCGR–
glucagon model, the corresponding pairs of residues, F6–Gln1421.40
and Y10–Tyr1381.36(one letter amino acid abbreviation is used to
designate glucagon residues), are located in close proximity and point
in the GLP1R–GLP1 complex12(Fig. 5b). The 12A˚distance between
L14–Trp295ECL2in the GCGR–glucagon model exceeds the range of
crosslinking distances in previous GLP1R–GLP1 models (8–9A˚)12,
though this may reflect differences between GCGR and GLP1R
There is no clear consensus on the binding site location of peptide
The GCGR–glucagon model illustrates a way to account for the
extensive interactions of the peptide with ECLs, as well as residues
reveals that some of the binding site residues previously positioned at
the top of 7TM helices or in ECLs30–32are in fact located deeper in the
residues allows glucagon to reach deep into the pocket. Our model of
glucagon incorporates a hypothetical N-capping conformation33of the
peptide helix in residues F6–T7–Y10, similar to theoneobserved inthe
receptor-bound PACAP26, though other conformations of this region
are supported by mutagenesis (Figs 4, 5c, d, Supplementary Table 5)
and photo-crosslinking studies on GCGR6,19,21,27,31,32,34and other class
B GPCRs8–13,20,29,30,35,36. In the 7TM domain, many residues predicted
to interact with glucagon show dramatic effects on glucagon binding
Figure 5 shows how these mutations line the 7TM binding site in the
GCGR–glucagon model and include residues that are located deep
in the pocket (Tyr1491.47, Val1912.64, Gln2323.37, Glu3626.53and
of glucagon deep intothe GCGR pocket, a region that couldbeequally
important for ligand binding as in class A GPCRs.
In the loop region, residues Arg201, Tyr202, Asp208 and Trp215
suggests that residues Trp295 and Asn298 directly interact with glu-
ing. Although mutations of Asp218 (ref. 21), Cys2243.29(refs 21, 27),
Arg2253.30, Lys2864.64, Glu290 and Cys294 (Fig. 4, Supplementary
act with glucagon in the model, but can play a role in stabilizing the
loop conformation compatible with glucagon binding. For example,
ECL2 and ECL1 are stabilized by a disulphide bridge between Cys294
and between Asp218 and Arg2253.30, respectively. Similarly, Arg378
is proposed to play a role in glucagon binding indirectly by stabilizing
theECL3 conformation,whileTrp3045.36stabilizesECL2atthe inter-
face between helices V and VI.
The GCGR–glucagon model presented in Fig. 5 is based on crystal-
tion binding studies (Fig. 4, Supplementary Table 5)6,19,21,27,31,32,34, and
thus provides comprehensive insight into recognition between the
native ligand and its receptor. The hypothetical model of the complex
can offer a useful platform for the design of biochemical and biophys-
ical experiments detailing the complex structure, as well as the design
of stabilized constructs that may lead to solution of the full-length
Related class B GPCRs
The GCGR–glucagon model can be informative for understanding
common features that determine ligand recognition of other class B
receptors. The GCGR mutation data (Fig. 4, Supplementary Table 5),
logous residues in other class B GPCRs. Supplementary Table 6 shows
> 10-fold reduction
< 4-fold reduction
R K F
H Q V
L L L
P A N
K E F
Q P S
W R D
D K Y
C W P D T
–11 –10 –9
Log [glucagon (M)]
Specific binding (%)
–11 –10 –9
Specific binding (%)
Log [glucagon (M)]
–11 –10 –9
Specific binding (%)
Log [glucagon (M)]
Figure 4 | Effects of mutation studies in GCGR
(purple), 4–10-fold (orange), and .10-fold (red)
changes of IC50values for glucagon binding with
receptor expression .30% of wild-type
(Supplementary Table 5). Mutation studies to
investigate peptide ligand binding have been
previously reported for several class B GPCRs
including GCGR6,19,21,27,31,32,34, GLP1R6,8,18,20,29,36,
GIPR9,37, rSCTR11,30,35,38,43andVPAC1 (refs39–41)
(Supplementary Table 6). The most conserved
residues in helices I to VII of class B GPCRs18are
boxed and shown in bold. b–d, Representative
binding curves of GCGR mutants with glucagon.
Data are expressed as a percentage of specific125I-
glucagon binding in the presence of 0.02 nM
unlabelled peptide. Each point (6s.e.m.)
represents the mean value of at least three
independent experiments done in triplicate (IC50
are shown in Supplementary Table 5).
2 5 J U LY 2 0 1 3 | V O L 4 9 9 | N A T U R E | 4 4 7
Macmillan Publishers Limited. All rights reserved
an overview of 274 previously reported mutants of GCGR6,19,21,27,31,32,34,
(also known as gastric inhibitory polypeptide receptor) (GIPR)9,37,
rat secretin receptor (rSCTR)11,30,35,38,43, and vasoactive intestinal pep-
tide and pituitary adenylate cyclase-activating polypeptide receptor 1
(VPAC1, also known as VIPR1) (refs 39–41). For example, mutations
of other class B GPCRs in residues that align to GCGR residues
Tyr65ECD(ref. 8), Tyr84ECD(ref. 8), Leu85ECD(ref. 8), Tyr1451.43
(ref. 35), Tyr1491.47(refs 29, 35), Lys1872.60(refs 9, 18, 20, 29, 30, 39),
Ile1942.67(refs 9, 18, 20, 29, 30, 39), Asp1952.68(refs 11, 20, 29, 30),
Leu1982.71(refs 11, 21), Arg2253.30(ref. 20), Gln2323.37(refs 9, 29),
Lys2864.64(ref. 36), Glu290ECL2(ref. 36), Trp295ECL2(refs 11, 36),
Asn298ECL2(refs 11, 36), Phe3656.56(refs 9, 11) and Leu3867.43(ref. 40)
have been shown to affect peptide ligand binding and/or potency,
model demonstratesthatresidues which have been identified to inter-
act with the homologous residues Q3 of glucagon, D3 of secretin, and
and VPAC1 (Arg1882.60and Lys1952.67) (ref. 39), respectively (Sup-
plementary Table 6).
The distinct structural features and larger binding pocket of the
GCGR 7TM domain provide new insights into the molecular details
of peptide ligand binding, and a more reliable structural template for
the design of specific and potent small molecules for the treatment of
type 2 diabetes. Moreover, the apparent overlap of class B GPCR
binding sites suggests that, despite possible structural differences
between class B GPCRs, the GCGR crystal structure might offer
new opportunities to construct structural models to describe interac-
tions between peptide ligands and other class B GPCRs. This is par-
ticularly exciting for those receptors involved in glucose regulation,
including GLP1R and GIPR.
BRIL–GCGR(DECD/DC) was expressed in Spodoptera frugiperda insect cells,
solubilizedwith 1/0.1% (w/v) of n-dodecyl-b-D-maltopyranoside and cholesteryl
hemisuccinate for 2h at 4uC, and purified by immobilized metal ion affinity
chromatography with 50–200mM of GCGR antagonist ligand NNC0640.
Protein at 80mgml21was mixed with monoolein and cholesterol in a ratio of
40%:54%:6% (w/w/w) to form lipidic cubic phase42, and crystallized in 100mM
MES pH6.0, 140–200 mM Na/K tartrate tetrahydrate, 9–17% (v/v) PEG 400,
0.35–0.55% (v/v) Jeffamine M-600, pH7.0, and 200 mM NNC0640 at 20uC.
X-ray data were collected on the 23ID-D beamline (GM/CA CAT) at the Advanced
(Supplementary Table 2). A single wavelength anomalous dispersion (SAD) data set
was collected at 4A˚from a single crystal soaked with tantalum bromide (Ta6Br12;
Supplementary Fig. 7). Phase information from the SAD data set confirmed the
diffraction data were collected from 14 crystals and anisotropically truncated before
refinement in a*, b*, and c* to 3.3, 3.4, and 3.3A˚, respectively. We report the final
structure at 3.4A˚resolution, and data collection, processing, structure solution and
refinement are described in the Methods.
The model of the GCGR–glucagon complex was constructed using the struc-
tures of the GCGR 7TM domain presented here, the GCGR ECD (PDB: 4ERS),
the GLP1R–GLP1 complex (PDB: 3IOL), and the N-capped conformation of
PACAP (PDB: 1GEA).
GCGR ECDGCGR ECD GCGR ECD
GCGR linker GCGR linker
GCGR ECL1GCGR ECL1
GCGR 7TMGCGR 7TM
F365 F365F365 F365
E362E362 E362 E362
K286K286 K286 K286
W295W295 L198W295W295 L198
Figure 5 | Model of GCGR bound to glucagon. a, b, GCGR with the ECD
(magenta) and 7TM domain (blue) bound to glucagon (green). Residues 122–
126 and 199–218 (brown) are not defined in the GCGR ECD (GCGR-linker)
(PDB: 4ERS) and 7TM domain (ECL1) crystal structures, respectively. The
GCGR ECD structure and the interactions between GCGR ECDand glucagon
resemble those in the GCGR ECD (PDB: 4ERS)6and GLP1–GLP1R-ECD
complex (PDB: 3IOL)8structures, respectively. c, d, The effects of mutation
the GCGR binding surface using the colour coding presented in Fig. 4.
pairs that are homologous to residue pairs identified in GLP1R–GLP1
crosslinking studies12are underlined.
4 4 8 | N A T U R E | V O L 4 9 9 | 2 5 J U LY 2 0 1 3
Macmillan Publishers Limited. All rights reserved
Binding studies were performed using transiently transfected CHO-K1 and
HEK293T cells. Either whole cells or prepared membranes were used to measure
binding affinity (IC50) of glucagon or NNC0640 using radiolabelled glucagon or
Full Methods and any associated references are available in the online version of
Received 7 March; accepted 17 June 2013.
Published online 17 July 2013.
and significance for drug discovery. Nature Rev. Drug Discov. 7, 339–357 (2008).
diabetes and obesity therapy. Pharmacol. Ther. 135, 247–278 (2012).
Katritch, V., Cherezov, V. & Stevens, R. C. Structure-function of the G protein-
Hoare, S. R. Mechanisms of peptide and nonpeptide ligand binding to class B
G-protein-coupled receptors. Drug Discov. Today 10, 417–427 (2005).
Pal,K.,Melcher,K.& Xu,H.E.Structureandmechanismforrecognition ofpeptide
hormones by Class B G-protein-coupled receptors. Acta Pharmacol. Sin. 33,
Koth, C. M. et al. Molecular basis for negative regulation of the glucagon receptor.
Proc. Natl Acad. Sci. USA 109, 14393–14398 (2012).
Parthier, C., Reedtz-Runge, S., Rudolph, R. & Stubbs, M. T. Passing the baton in
class B GPCRs: peptide hormone activation via helix induction? Trends Biochem.
Sci. 34, 303–310 (2009).
the extracellular domain of the glucagon-like peptide-1 receptor. J. Biol. Chem.
285, 723–730 (2010).
natural ligand. Mol. Pharmacol. 77, 547–558 (2010).
10. Miller, L. J., Dong, M., Harikumar, K. G. & Gao, F. Structural basis of natural ligand
binding and activation of the Class II G-protein-coupled secretin receptor.
Biochem. Soc. Trans. 35, 709–712 (2007).
11. Dong, M. et al. Mapping spatial approximations between the amino terminus of
FASEB J. 26, 5092–5105 (2012).
12. Miller, L. J. et al. Refinement of glucagon-like peptide 1 docking to its intact
receptor using mid-region photolabile probes and molecular modeling. J. Biol.
Chem. 286, 15895–15907 (2011).
13. Dong, M. et al. Molecular basis of secretin docking to its intact receptor using
multiple photolabile probes distributed throughout the pharmacophore. J. Biol.
Chem. 286, 23888–23899 (2011).
for residue 19 of parathyroid hormone (PTH) and PTH-related protein analogs in
transmembrane domain two of the type 1 PTH receptor. Mol. Endocrinol. 17,
15. de Graaf, C., Rein, C., Piwnica, D., Giordanetto, F. & Rognan, D. Structure-based
discovery of allosteric modulators of two related class B G-protein-coupled
receptors. ChemMedChem 6, 2159–2169 (2011).
16. Chun,E.etal.Fusionpartnertoolchest for thestabilizationandcrystallizationofG
protein-coupled receptors. Structure 20, 967–976 (2012).
C.) 366–428 (Academic Press, 1995).
18. Wootten, D., Simms, J., Miller, L. J., Christopoulos, A. & Sexton, P. M. Polar
transmembrane interactions drive formation of ligand-specific and signal
pathway-biased family B G protein-coupled receptor conformations. Proc. Natl
Acad. Sci. USA 110, 5211–5216 (2013).
19. Unson, C. G. et al. Rolesof specificextracellular domains of the glucagon receptor
in ligand binding and signaling. Biochemistry 41, 11795–11803 (2002).
20. Xiao, Q., Jeng, W. & Wheeler, M. B. Characterization of glucagon-like peptide-1
receptor-binding determinants. J. Mol. Endocrinol. 25, 321–335 (2000).
21. Roberts, D. J., Vertongen, P. & Waelbroeck, M. Analysis of the glucagon receptor
first extracellular loop by the substituted cysteine accessibility method. Peptides
32, 1593–1599 (2011).
22. Wu, H. et al. Structure of the human k-opioid receptor in complex with JDTic.
Nature 485, 327–332 (2012).
23. Manglik, A. et al. Crystal structure of the m-opioid receptor bound to a morphinan
antagonist. Nature 485, 321–326 (2012).
24. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. & Schioth, H. B. The G-protein-
coupled receptors in the human genome form five main families. Phylogenetic
analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272
25. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors.
Nature 494, 185–194 (2013).
26. Inooka, H. et al. Conformation of a peptide ligand bound to its G-protein coupled
receptor. Nature Struct. Biol. 8, 161–165 (2001).
27. Pre ´vost, M. et al. Mutational and cysteine scanning analysis of the glucagon
receptor N-terminal domain. J. Biol. Chem. 285, 30951–30958 (2010).
28. Ahn,J.M.,Medeiros,M.,Trivedi,D.& Hruby,V.J.Development ofpotenttruncated
glucagon antagonists. J. Med. Chem. 44, 1372–1379 (2001).
29. Coopman, K. et al. Residues within the transmembrane domain of the glucagon-
like peptide-1 receptor involved in ligand binding and receptor activation:
modelling the ligand-bound receptor. Mol. Endocrinol. 25, 1804–1818 (2011).
30. Di Paolo, E. et al. Contribution of the second transmembrane helix of the secretin
receptor to the positioning of secretin. FEBS Lett. 424, 207–210 (1998).
31. Perret, J. et al. Mutational analysis of the glucagon receptor: similarities with the
vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide
(PACAP)/secretin receptors for recognition of the ligand’s third residue. Biochem.
J. 362, 389–394 (2002).
32. Runge, S. et al. Three distinct epitopes on the extracellular face of the glucagon
33. Neumann, J. M. et al. Class-B GPCR activation: is ligand helix-capping the key?
Trends Biochem. Sci. 33, 314–319 (2008).
34. Cascieri, M. A. et al. Characterization of a novel, non-peptidyl antagonist of the
human glucagon receptor. J. Biol. Chem. 274, 8694–8697 (1999).
35. Di Paolo, E. et al. Mutations of aromatic residues in the first transmembrane helix
36. Koole, C. et al. Second extracellular loop of human glucagon-like peptide-1
receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor
activation. J. Biol. Chem. 287, 3642–3658 (2012).
37. Tseng, C. C. & Lin, L. A point mutation in the glucose-dependent insulinotropic
peptide receptor confers constitutive activity. Biochem. Biophys. Res. Commun.
232, 96–100 (1997).
38. Ganguli, S. C. et al. Protean effects of a natural peptide agonist of the G protein-
coupled secretin receptor demonstrated by receptor mutagenesis. J. Pharmacol.
Exp. Ther. 286, 593–598 (1998).
39. Solano, R. M. et al. Two basic residues of the h-VPAC1 receptor second
transmembrane helix are essential for ligand binding and signal transduction.
J. Biol. Chem. 276, 1084–1088 (2001).
40. Ceraudo, E. et al. Spatial proximity between the VPAC1 receptor and the amino
terminus of agonist and antagonist peptides reveals distinct sites of interaction.
FASEB J. 26, 2060–2071 (2012).
41. Tan, Y. V., Couvineau, A. & Laburthe, M. Diffuse pharmacophoric domains of
vasoactive intestinal peptide (VIP) and further insights into the interaction of VIP
with the N-terminal ectodomain of human VPAC1 receptor by photoaffinity
labeling with [Bpa6]-VIP. J. Biol. Chem. 279, 38889–38894 (2004).
42. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic
mesophases. Nature Protocols 4, 706–731 (2009).
polypeptide/secretin family of receptors on the secretin receptor functionality.
Peptides 20, 1187–1193 (1999).
Acids Res. 40, D370–D376 (2012).
Supplementary Information is available in the online version of the paper.
for technology development (V.C. and R.C.S.), and PSI:Biology grant U54 GM094618
PSI:Biology grant U54 GM094586 for structure QC; The Ministry of Health grants
2012ZX09304-011 and 2013ZX09507002 (M.-W.W.), Shanghai Science and
Technology Development Fund 11DZ2292200 (M.-W.W.); Novo Nordisk-Chinese
Academy of Sciences Research Fund NNCAS-2011-7 (M.-W.W.); Thousand Talents
Program in China (R.C.S. and M.-W.W.); NIH Postdoctoral Training Grant (NRSA) F32
DK088392 (F.Y.S.); The Netherlands Organization for Scientific Research (NWO)
through a VENI grant (Grant 700.59.408 to C.d.G.); COST Action CM1207, GLISTEN
(C.d.G). We also thank V. Hruby and M. Cai for advice with the glucagon binding assay
and general discussions; J. Velasquez for help with molecular biology; T. Trinh and
M. Chu for help with baculovirus expression; K. Kadyshevskaya for assistance with
figure preparation; X. Q. Cai, J.Wang, Y. Feng, A. T. Dai, Y. Zhou, J.J. Deng, Y. B. Dai and
J. W. Zhao for technical assistance in mutation studies; A. Walker for assistance with
manuscript preparation; and J. Smith and R. Fischetti for assistance in development
and use of the minibeam and beamtime at GM/CA-CAT beamline 23-ID at the
Advanced Photon Source, which is supported by National Cancer Institute grant
Y1-CO-1020 and National Institute of General Medical Sciences grant Y1-GM-1104.
Author Contributions F.Y.S. designed, expressed, characterized and screened
LCP, optimized crystallization conditions, grew crystals, collected diffraction data and
prepared the manuscript. G.W.H. and Q.X. solved and refined the structure, and
prepared the manuscript. V.C. collected and processed diffraction data, and prepared
the manuscript. M.H., D.Y., Z.Z. and C.Z. expressed the receptor, and performed the
mutagenesis and ligand-binding assay. V.K. and C.d.G. designed and analysed the
receptor mutagenesis studies, constructed the receptor–ligand model and prepared
the manuscript. D.W. and J.S.J. collected and processed SAD data and determined an
initial electron density map from experimental phases. W.L. and V.C. trained and
assisted in LCP crystallization. J.L. provided ligands for GCGR and prepared the
manuscript. R.C.S., F.Y.S., M.-W.W., V.K., V.C. and C.d.G. were responsible for the overall
project strategy and management and wrote the manuscript.
Author Information The coordinates and the structure factors have been deposited in
the Protein Data Bank under the accession code 4L6R. Reprints and permissions
information is available at www.nature.com/reprints. The authors declare no
competing financial interests. Readers are welcome to commenton the online version
(email@example.com) or M.-W.W. (firstname.lastname@example.org).
2 5 J U LY 2 0 1 3 | V O L 4 9 9 | N A T U R E | 4 4 9
Macmillan Publishers Limited. All rights reserved
BRIL–GCGR(DECD/DC) construct design and Sf9 expression. The human
wild-type GCGR DNA was synthesized by DNA 2.0 and codon optimized for
expression in Spodoptera frugiperda (Sf9) insect cells. The BRIL–GCGR(DECD/
thermally stabilized apocytochrome b562RIL (M7W, H102I, R106L) (referred to
as BRIL) from E. coli at residue 123 (ref. 16), and truncating the C terminus at
This chimaeric construct was obtained after screening 60 constructs of different
BRIL junction and C-terminal truncation sites to generate crystals with diffrac-
tion data of the highest quality and resolution. The construct was cloned into a
modified pFastBac1 vector (Invitrogen) containing an expression cassette with
haemagglutinin signal sequence at the N terminus, and a PreScission protease
site, 103His, and Flag tag at the C terminus. The BRIL–GCGR(DECD/DC)
fusion construct was expressed in Sf9 cells using the Bac-to-Bac baculovirus
expression system as described previously45. Sf9 cells at a density of 23106–
33106cells ml21were infected with P1 or P2 virus at a multiplicity of infection
(m.o.i.) of 7.5. Cells were harvested less than 48h post-infection and cell pellets
were stored at 280uC until used.
BRIL–GCGR(DECD/DC) fusion construct purification. Sf9 membranes
were prepared with 1 wash cycle of hypotonic buffer (25mM HEPES, pH7.5,
10mM MgCl2, 20mM KCl) in the presence of EDTA-free protease inhibitor
cocktail tablets (Roche) and 4 wash cycles of high-salt buffer (25mM HEPES,
pH7.5, 1M NaCl, 10mM MgCl2, 20mM KCl). Membrane pellets were homo-
genized in storage buffer (25mM HEPES, pH7.5, 500mM NaCl, 40% glycerol),
flash frozen in liquid nitrogen and stored at 280uC until use.
The GCGRantagonistligand NNC0640(Supplementary Fig. 1b) was essential
for purification and crystallization of the BRIL–GCGR(DECD/DC) fusion con-
struct. Two grams of washed membranes containing the BRIL–GCGR(DECD/
DC) fusion construct were resuspended in 30ml of buffer (25mM HEPES,
NNC0640 for 30min at room temperature. The receptor was solubilized with
1/0.1% (w/v) of n-dodecyl-b-D-maltopyranoside (Anatrace) and cholesteryl
hemisuccinate (Sigma) (DDM/CHS) for 2h at 4uC. The insoluble material was
pelleted by ultracentrifugation in a Ti70 rotor at 504,300g for 30min at 4uC. The
NaCl and DDM/CHS concentrations of the supernatant were adjusted to
800mM and 0.5/0.05%, respectively, by adding equal volume of talon binding
buffer (25mM HEPES, pH7.0, 1.475M NaCl, 10% glycerol). Protein was bound
the presence of 15mM imidazole, pH7.5, and 100mM NNC0640.
pH7.0, 800mM NaCl, 10% glycerol, 0.04/0.008% DDM/CHS, 30mM NNC0640,
glycerol, 0.02/0.004% DDM/CHS, 30mM NNC0640). The BRIL–GCGR(DECD/
DC) fusion construct was eluted with 2.5ml of elution buffer (25mM HEPES,
pH7.0, 150mM NaCl, 10% glycerol, 0.02/0.004% DDM/CHS, 30mM NNC0640,
zole. His-tagged PreScission protease was added to the samples and incubated
overnight at 4uC to remove the C-terminal 103His and Flag tags. Reverse talon
construct by flowing the sample through 200ml talon superflow resin twice. The
flow-through material, containing the cleaved BRIL–GCGR(DECD/DC) fusion
construct, was concentrated to 80mgml21using a Vivaspin centrifuge concen-
trator (GE Healthcare) with a 100 kilodalton (kDa) molecular weight cut-off.
BRIL–GCGR(DECD/DC) fusion construct lipidic cubic phase (LCP) crystal-
lization. For LCP crystallization, the BRIL–GCGR(DECD/DC) construct was
mixed with molten lipid at a ratio of 40/60% (v/v) using a mechanical syringe
mixer42. Due to the high detergent concentration, 10–15% (volume of LCP) of
5M NaCl was added after the lipid and protein were mixed to convert a desta-
bilized lipidic mesophase into LCP46. The host lipid for the LCP reconstitution
was monoolein (Sigma) with 10% (w/w) of cholesterol (AvantiPolar Lipids).
Crystallization trials were set up as previously described47. LCP-FRAP was used
to identify initial crystallization conditions that led to GCGR crystals48. Crystals
were obtained at 20uC in 100mM MES, pH6.0, 140–200mM Na/K tartrate
tetrahydrate, 9–17% (v/v) PEG 400, 0.35–0.55% (v/v) Jeffamine M-600, pH7.0,
about 5days (Supplementary Fig. 3); and harvested from the LCP matrix using
50mm MiTeGen micromounts and immediately flash frozen in liquid nitrogen.
(GM/CA CAT) at the Advanced PhotonSource(Argonne, Illinois) using a 20-mm
other GPCR structures49. Typically, 10–15 frames at 1u oscillation and 1–2s expo-
sure with non-attenuated beam were collected per crystal due to the fast onset of
radiation damage. A 93.9% complete at 3.3A˚data set was obtained by indexing,
Analysis of the final data set by the UCLA diffraction anisotropy server (http://
services.mbi.ucla.edu/anisoscale/) indicated that diffraction along the b* axis was
weaker than in the other two directions; therefore, reflections were subjected to a
and c*, respectively, before using them in the refinement.
Experimental phasing.Initialattemptsto find a molecularreplacementsolution
using previous class A GPCR structures as search models in Phaser51did not
generate any reliable solutions. Therefore, experimental phasing for the BRIL–
GCGR(DECD/DC) fusion construct was attempted by soaking the crystals with
from one crystal that was soaked overnight with 100mM tantalum bromide
line (GM/CA CAT) at the Advanced Photon Source using the peak wavelength
from the tantalum L3 edge (9.880keV). A beam size of 10mm with 53 attenu-
ation with 1u oscillation and 1s exposure per frame was used. A complete 360u
data set was acquired from a single crystal by collecting wedges of 30u with direct
portionofthe crystalforeachwedge.TheSADdata setwasintegratedand scaled
at 4A˚resolution using HKL2000. PHENIX.AutoSol52was used to search for the
yielding initial electron density maps.
generated based on all known GPCR structures using PHENIX.ROSETTA53. The
were then carried out using these GPCR models along with the high resolution
BRIL structure (PDB: 1M6T) as search models on a linux cluster54. The search
This MR solution was validated by the experimental phasing maps (Supplementary
Fig. 8), and by the appearance of density not present in the search model. The
experimental SIRAS phases calculated from the heavy atom were good up to 6–7A˚.
(ref. 55), autoBUSTER56(Buster v2.8.0), and PHENIX.AutoBuild57, followed by
manualexamination andrebuildingofrefinedcoordinates in COOT58usingboth
procedure59(Supplementary Fig. 9). We state 3.4A˚as the overall effective resolu-
tion of this structure; however, data to 3.3A˚were included in refinement, which
improved the R/Rfreestatistics (Supplementary Table 2).
At 3.4A˚resolution, the electron densities for the majority of residues in the
GCGR 7TM structure are visible, except for residues Arg201–Trp215 (corres-
ponding to ECL1), and therefore these residues were not built into the GCGR
Phe365–Glu371 were built into the GCGR structure, but they contained breaks
in the electron densities of the Cabackbone. Hence, other conformations are
possible for these residues.
Although we do not observe density for NNC0640 in the canonical ligand
binding pocket, this ligand is required to obtain diffraction quality crystals of
the BRIL–GCGR(DECD/DC) construct. There are two electron density blobs
outside of canonical ligand-binding pocket, one at the bottom of helix VI and
VII near Lys3496.40, and the other at helix I near Trp1451.43. However, both of
them are too small to accommodate NNC0640.
Energy-based conformational modelling of the GCGR–glucagon complex.
Glucagon was docked into the crystal structure of the GCGR ECD (PDB: 4ERS,
residues 28–123) (ref. 6) using the crystal structure of the closely related GLP1R–
GLP1 complex (PDB: 3IOL)8as a template. All molecular modelling and docking
was performed using ICM molecular modelling software60(v. 3.7). The initial
a-helical conformation of glucagon peptide residues 11–29 was modelled based
on GLP1 residues 17–35, and soft tethers between corresponding backbone Ca
and the interacting side chains in the ECD binding pocket were optimized (3 inde-
pendent simulations of 106steps) using ICM global optimization procedure in
internal coordinates60,61with improved conformational energy terms for protein
and peptides4and ‘tether weight’50.1.
Macmillan Publishers Limited. All rights reserved
This flexible energy-based docking/optimization procedure involved all torsion Download full-text
coordinates in the regions that are not defined by crystal structures, including
stalk region (125–136), ECL2 region (289–310), ECL3 (368–377), as well as 31
other residues lining the 7TM binding pocket.
The following three soft harmonic restraints derived from experimental
crosslinking data in GLP1R and GLP1 (ref. 12) were applied between gluca-
gon and GCGR side chains to guide docking: F6(cb)–Gln1421.40(cd), Y10(cb)–
Tyr1381.36(oh), L14(cb)–Trp295ECL2(ch2). Two intramolecular harmonic
restraints were also applied to glucagon residues, T7(og1)–Y10(n) and F6(cz)–
Y10(cz), to facilitate N-capped formation in glucagon, as suggested by previous
comparative studies of class B peptide ligands33. Finally, a restraint was applied
between the positively charged N-terminal amino group of glucagon and the
carboxyl group of Glu3626.53. The importance of the carboxyl group of
Glu3626.53, which is the only negatively charged residue in the 7TM binding
pocket, is supported by GCGR (Supplementary Table 5) and GLP1R (ref. 29)
mutation studies (Supplementary Table 6). As the N terminus is the only basic
moietyin thefirst 10 residuesofglucagon,apotentialGlu3626.53saltbridgewith
Monte-Carlo global optimization, and 455 were locally minimized in the course
of this procedure. The special ‘local’ sampling option was applied to the ECL1
region backbone to allow efficient optimization. Three independent runs of the
global optimization procedure (107steps each) resulted in similar best energy
conformations within 2.5A˚r.m.s.d. for the glucagon peptide non-hydrogen
flexible, sampling multiple orientations relative to the 7TM domain6. The model
also does not attempt to infer a specific functional state of the receptor, partially
because such a state is not precisely defined for the 7TM crystal structure itself.
For instance, NNC0640 used to stabilize the 7TM receptor fragment is a com-
petitive antagonist to glucagon, which may have an effect on the crystallized
conformation, even though NNC0640 is absent in the final structure. The accu-
racy of the GCGR–glucagon model may also be limited by the weak electron
density of ECL2 and the top of helix V (residues 289–310), and the assumption
that glucagon binds GCGR in an N-capped conformation7,33.
(cDNA) encoding the human GCGR was originally obtained from GeneCopoeia
and cloned into the expression vector pcDNA3.1/V5-His-TOPO (Invitrogen) at
the HindIII and EcoRI sites. The single and double mutants were constructed by
PCR-based site directed mutagenesis. CHO-K1 cells were seeded onto 96-well
poly-D-lysine treated cell culture plates (PerkinElmer) at a density of 2.73104
cells per well. After overnight culture, the cells were transiently transfected with
wild-type or mutant GCGR DNA using Lipofectamine 2000 transfection reagent
HEPES, pH7.4, and 0.1% bovine serum albumin (BSA)) for 2h at 37uC. For
homogeneous binding, the cells were incubated in binding buffer with constant
concentration of125I-glucagon (40pM) and different concentrations of unla-
belled glucagon (0.02nM to5mM) at room temperature for 3h. Cells were
washed three times with ice-cold PBS and lysed by 50ml lysis buffer (PBS sup-
plemented with 20mM Tris-HCl, 1% Triton X-100, pH7.4). The plates were
subsequently counted for radioactivity (counts per minute, CPM) in a scintil-
lation counter (MicroBeta2 Plate Counter, PerkinElmer) using a scintillation
cocktail (OptiPhase SuperMix, PerkinElmer). Specific binding was determined
by subtracting non-specific binding observed in the presence of 5mM unlabelled
primary antibody (anti-GCGR, Epitomics) at room temperature for 1h. The cells
were then washed three times with PBS containing 1% BSA followed by a 1h
incubation with anti-rabbit Alexa-488-conjugated secondary antibody (1:300,
Invitrogen) at 4uC in the dark. After washes, the cells were resuspended in
200ml of PBS containing 1% BSA for detection in a flow cytometer (Accuri C6,
BD Biosciences) using laser excitation and emission wavelengths of 488 and
were collected and fluorescence intensity of positive expression cell population
NNC0640 binding assay (cell membrane based binding). NNC0640 binding
expressing GCGR constructs. Approximately 1.23108transfected HEK293T
cells were harvested, suspended in 10ml ice-cold membrane binding buffer
(25mM Tris-HCl, 0.1% BSA and 1mM EDTA, pH 7.4) and centrifuged for
5min at 200g. The resulting pellet was resuspended in cold membrane binding
at 20,000g. The precipitate containing the plasma membranes was suspended in
membrane binding buffer containing protease inhibitor (Sigma-Aldrich) and
stored at 280uC. Protein concentration was determined using a protein BCA
assay kit (Pierce Biotechnology).
For homogeneous binding, cell membrane homogenates (20mg protein per
well) were incubated in membrane binding buffer with constant concentration
of3H-NNC0640 (50nM, labelled by PerkinElmer) and serial dilutions of unla-
belled NNC0640 (1.26nM to 100mM) at room temperature for 5h. Nonspecific
bindingwas determined inthe presenceof100mMNNC0640.Followingincuba-
tion, the samples were filtered rapidly in vacuum through glass fibre filter plates
(Millipore). After soaking and rinsing 4 times with ice-cold binding buffer, the
filters were dried and counted for radioactivity in a scintillation counter
Western blot. Protein samples were prepared as above, separated by 10% SDS–
PAGE and transferred to nitrocellulose membranes. After a 2h incubation with
blocking buffer, the membranes were incubated with 1:1,000 primary antibody
(anti-V5, Sigma) overnight. The membranes were then washed three times with
TBS-T buffer (0.05M Tris, 0.15M NaCl, 0.1% (v/v) Tween) followed by a 2h
(1:1,000, Cell Signaling Technology). The membranes were washed again and then
manufacturer’s instructions. Each membrane was exposed to X-ray film for detect-
ing the blots. Bands were quantified with Quantity One Software (Bio-Rad).
Statistical analysis. Results are presented as means6s.e.m. Changes in specific
radiolabelled ligands binding and cell surface expression of GCGR constructs
were normalized to those measured with wild-type GCGR control (100%). IC50
values in binding assay were determined by nonlinear regression analysis using
the Prism 5 software (GraphPad Software).
by coupling cell-surface detection with small-scale parallel expression. Protein
Expr. Purif. 56, 85–92 (2007).
46. Misquitta, Y. & Caffrey, M. Detergents destabilize the cubic phase of monoolein:
47. Cherezov, V., Peddi,A.,Muthusubramaniam,L., Zheng,Y.F.& Caffrey,M.Arobotic
system for crystallizing membrane and soluble proteins in lipidic mesophases.
Acta Crystallogr. D 60, 1795–1807 (2004).
48. Xu, F., Liu, W., Hanson, M. A., Stevens, R. C. & Cherezov, V. Development of an
automated high throughput LCP-FRAP assay to guide membrane protein
crystallization in lipid mesophases. Cryst. Growth Des. 11, 1193–1201 (2011).
49. Cherezov, V. et al. Rastering strategy for screening and centring of microcrystal
samples of human membrane proteins with a sub-10mm size X-ray synchrotron
beam. J. R. Soc. Interface 6 (Suppl 5), S587–S597 (2009).
50. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol. 276, 307–326 (1997).
of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D 65, 582–601 (2009).
53. Terwilliger, T. C. et al. phenix.mr_rosetta: molecular replacement and model
rebuilding with Phenix and Rosetta. J. Struct. Funct. Genomics 13, 81–90 (2012).
54. Schwarzenbacher, R., Godzik, A. & Jaroszewski, L. The JCSG MR pipeline:
optimized alignments, multiple models and parallel searches. Acta Crystallogr.
D 64, 133–140 (2008).
by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).
56. BUSTER v. 2.8.0 (Global Phasing, Cambridge, UK 2009).
57. Terwilliger, T. C. et al. Iterative model building, structure refinement and density
58. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of
Coot. Acta Crystallogr. D 66, 486–501 (2010).
59. Bhat, T. Calculation of an OMIT map. J. Appl. Crystallogr. 21, 279–281 (1988).
60. ICM Manual v. 3.0 (MolSoft, La Jolla, California 2012).
61. Arnautova, Y. A., Abagyan,R. A. & Totrov, M. Development of a new physics-based
internal coordinate mechanics force field and its application to protein loop
modeling. Proteins 79, 477–498 (2011).
Macmillan Publishers Limited. All rights reserved