METHODS AND APPLICATIONSApplication of the NZ-1 Fab as a
crystallization chaperone for PA
tag-inserted target proteins

Risako Tamura,1 Rika Oi,1 Satoko Akashi,1 Mika K. Kaneko,2 Yukinari Kato,2 andTerukazu Nogi 1*
1

Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan
Tohoku University Graduate School of Medicine, Tohoku, Japan

2

Received 27 September 2018; Accepted 15 January 2019
DOI: 10.1002/pro.3580
Published online 21 January 2019 proteinscience.org

Abstract: An antibody fragment that recognizes the tertiary structure of a target protein with highafﬁnity can be utilized as a crystallization chaperone. Difﬁculties in establishing conformation-speciﬁc
antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we
attempted to establish an alternative method to promote the crystallization of target proteins using an
already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with anextremely high afﬁnity. It was also established that the PA tag is accommodated in the antigen-binding
pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target
protein displaying a PA tag. Speciﬁcally, we inserted the PA tag into the β-hairpins of the PDZ tandem
fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the
NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initialinsertion designs produced only moderate-resolution structures, eliminating the solvent-accessible
space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably.
Our results demonstrate that the NZ-1-PA system efﬁciently promotes crystallization of the target
protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because
the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.
Keywords: crystallization chaperone; protein crystallography; monoclonal antibody; antigen-binding
fragment; epitope tag insertion

Introduction
Additional Supporting Information may be found in the online
version of this article.
Grant sponsor: Japan Agency for Medical Research and Development JP16am0101020 JP17am0101078 JP17am0101078;
Grant sponsor: Japan Society for the Promotion of Science
JP17K19206 JP26291016.
*Correspondence to: Terukazu Nogi, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
E-mail: nogi@yokohama-cu.ac.jp

Published by Wiley © 2019 The Protein Society

Antibody fragments such as antigen-binding fragments(Fab) and variable fragments (Fv) can serve as crystallization chaperones in protein crystallography.1–4 Antibody fragments increase the probability of forminginter-molecular contacts required for lattice formation.
This strategy is particularly important for difﬁcultto-crystallize proteins such as membrane proteins covered with ﬂuidic detergent micelles and glycoproteins

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containing ﬂexible sugar moieties. Although various
immunization and screening techniques have beendeveloped,5–7 the establishment of antibodies that promote co-crystallization is still a difﬁcult task. We,
therefore, attempted to explore an alternative strategy
to promote crystallization by inserting the epitope
sequence of an established antibody into target proteins and subsequently preparing complexes with antibody fragments. Speciﬁcally, we utilized a monoclonal
antibody NZ-1 and its epitope tag. NZ-1 was originallygenerated by immunizing rats with the plateletaggregation-stimulating domain of human podoplainin
and the epitope was identiﬁed as the tetradecapeptide
PA14 (EGGVAMPGAEDDVV).8 Although the central
portion, PA10 (GVAMPGAEDD), is indispensable andsufﬁcient for speciﬁc binding with NZ-1,9 PA14 andPA12 (GVAMPGAEDDVV) both show exceptionallyhigh afﬁnity and slow dissociation from NZ-1.10 Due tothese properties, the PA12 epitope has been developed
as the PA tag for protein puriﬁcation. In addition, acrystallographic analysis of the NZ-1 Fab–PA14 peptide complex has shown that the epitope binds to NZ-1
in a bent conformation with a Type II β-turn between
Pro-5 and Gly-6, making it possible to insert a PA tag
into the loop regions of target proteins.11 In fact, a PAinsertion has already been used as a probe to detect
conformational states of a modular cell-surface receptor
by comparing the accessibilities of the PA tag to NZ-1.11
NZ-1 labeling of an inserted PA tag is also useful for
locating subunits or domains within macromolecularcomplexes by electron microscopy (EM) imaging.12,13 In
this study, we further examined the applicability of theinserted PA tag-NZ-1 Fab pair in generating welldiffracting crystals of target proteins.
As a target for PA-insertion, we selected the periplasmic soluble fragment from a Site-2 protease(S2P) homolog from the hyper-thermophilic bacterium Aquifex (A.) aeolicus.14 This soluble fragment
contains two tandemly arranged PSD95/Dlg/ZO-1(A) 264 - 265

182 -183(PDZ) domains (hereafter, the soluble fragment isreferred to as the PDZ tandem). In our previous work,
we obtained crystals of the PDZ tandem fragment,
which diffracted X-rays up to 2.8 Å resolution.15 We
found that the PDZ tandem was loosely packed in the
crystal with a relatively high solvent content (74%),
which is probably due to the abundance of ﬂexible
lysine residues on the molecular surface. In thatstudy, we generated a monoclonal antibody against
the PDZ tandem to utilize the Fab fragment as the
crystallization chaperone. As a result, we improved
the crystallographic resolution to 2.2 Å and determined the crystal structure more precisely.15 The successful previous application of the standard approachfor chaperone-assisted crystallography makes the
PDZ tandem a good system to test and evaluate anovel approach.
Here, we tested if we could insert the PA tag into
the PDZ tandem without disrupting the tertiary
structure and improve the resolution by using theNZ-1 Fab as the crystallization chaperone.

Results
Insertion of the PA tag into β-hairpins andstructure determination of the Fab-bound form
The canonical PDZ domain consists of six β-strands,
βA-F, and two α-helices, αA–B. Both of the two PDZ
domains (PDZ-N and -C) forming the PDZ tandem
are circular permutants of the canonical PDZ-fold(Fig. 1). As a result, the non-canonical PDZ domainsof the PDZ tandem possess the N-terminus before βC
and the C-terminus after βB while βF and βA are connected by a loop. We selected these two βF-βA loops of
PDZ-N and -C as the PA-insertion sites (Fig. 1(A))
because both of the two loops protrude outside of the
PDZ tandem fragment. In the present work, we specify the respective PDZ tandem mutants based on theresidues ﬂanking the PA tag sequence and the length

(B)

B

N

C

E

C

B

D

F

A

A

βF-βA loop
PDZ-C

PDZ-N

Figure 1. 3D structure of the PDZ tandem from the A. aeolicus Site-2 protease homolog. (A) Ribbon model of PDZ tandem. The
six β-strands of the respective PDZ domains are colored differently where PDZ-N and PDZ-C are shown in bright and pale colors,
respectively. The deleted loop residues for the PA-insertion in the β-hairpins are indicated in magenta with dotted circles.
(B) Topology diagram of the circular-permutant PDZ domain. The loop connecting the βF and βA strands is colored in magenta
and indicated with a dotted circle. In the present work, the PA tag was inserted here.

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Crystallizaion of PA-inserted target with NZ-1 Fabof the insertion. For instance, the mutant with the
PA12 sequence inserted between Arg-181 and Glu184 in the PDZ-N is termed as PDZ tandem(181-PA12-184). Similarly, the PDZ-C mutant with
the PA-insertion is termed as PDZ tandem(263-PA12-266).
The two PDZ tandem mutants were produced as
glutathione-S-transferase (GST)-fusion proteins. The
mutant fragments with the PA-insertion were monodisperse after removal of the GST portion. In sizeexclusion chromatography, both of the mutants
eluted at elution volumes comparable to that of thewild type (Supporting Information Fig. S1(A–C)),
indicating that the PA insertion did not affect thefolding of the PDZ domains. As a control, we introduced an L259R mutation to PDZ tandem (263PA12-266). Leu-259 participates in the hydrophobic
core and contributes to the structural integrity of
the PDZ-C domain. The mutation was therefore
expected to cause partial unfolding of the PDZ tandem fragment. As expected, we observed a signiﬁcantpeak shift to a smaller elution volume than that ofwild type in the size-exclusion chromatography(Supporting Information Fig. S1(D)). The peak shifts
observed in the PDZ tandem (181-PA12-184) and(263-PA12-266) were negligible compared with thatobserved in the L259R mutant, which is supportive of
the correct folding for both of the PDZ tandem(181-PA12-184) and (263-PA12-266). Subsequently,
we prepared complexes with the NZ-1 Fab. Bothmutants bound stably to the NZ-1 Fab, and almost no
dissociation was observed during fractionation bysize-exclusion chromatography. After puriﬁcation, we
attempted to crystallize the mutants alone and incomplex with the NZ-1 Fab. However, we obtained no
crystals of the solitary mutants at least within the
search space of our screening. As mentioned above,
the wild-type PDZ tandem tends to crystallize
through loose lattice contacts. Both of the PAinsertion sites were involved in lattice contacts forthe wild-type crystal, so there is a possibility that theinsertions disrupt that form.
Although the mutants seemed to become lesscrystallizable, we discovered several crystallization
conditions for both of the two mutants in complex
with the NZ-1 Fab. Many poorly diffracting cocrystals were screened before we selected crystals
that allowed us to determine crystal structures to resolutions of 3.2 Å and 4.0 Å for PDZ tandems(181-PA12-184) and (263-PA12-266) in complex with
the NZ-1 Fab, respectively (Figs. 2(A–C) and 3(A–C)).
Inspection of the crystal packing indicated that not
only the NZ-1 Fab but also the PDZ tandem substantially contributed to the lattice formation in bothcases. In particular, the moderate resolution of theco-crystal with PDZ tandem (181-PA12-184) was
probably due to loose lattice contacts. In the course ofmodel building, we observed weak and disordered

Tamura et al.

electron densities for the PDZ-C portion. PDZ-C isconnected with the PA-inserted PDZ-N domain
through a short linker, and the PDZ tandem, therefore, showed conformational ﬂexibility. The weakelectron densities suggested that PDZ-C was involvedin the lattice contacts to some extent, but we could
not assign a reliable model for this portion in the end[Fig. 2(A)].

Structural details of PDZ tandem (181-PA12-184)in the Fab-bound form
In the NZ-1 Fab-PDZ tandem (181-PA12-184) complex, the interaction mode of the complementarity
determining regions with the inserted PA tag wasessentially consistent with that observed in the NZ-1
Fab-PA14 co-crystal, excepting the involvement of ametal ion [Fig. 4(A, B)]. The electron density indicated that a metal ion is co-ordinated by Glu-8 andAsp-9 from the PA tag together with Asp-75 from the
NZ-1 heavy chain, although these two PA residues
were recognized by Lys-76 of the heavy chain in the
NZ-1 Fab-PA14 complex [Supporting Information
Fig. S2(A)]. The metal ion is presumed to be a cadmium ion present in the crystallization buffer. However, we could not determine the identities of theresidual electron densities due to the absence of
anomalous data near the absorption edge. Likewise,
coordination geometry, such as monodentate orbidentate co-ordination by carboxyl groups, could not
be unambiguously assigned due to the poor quality ofelectron density map.
As the NZ-1 Fab rigidiﬁes the main chain conformation of the PA tag when it is accommodated into
the antigen-binding pocket, the Cα atoms of Gly-1 andVal-12 were separated by 14.1 Å, which is comparable
to the 12.4 Å distance in the NZ-1 Fab-PA14 co-crystal[Fig. 4(A,B)]. The separation of these two residues
caused signiﬁcant conformational changes in the target PDZ-N domain at the junction with the PA tag.
For instance, Glu-184 and Val-185 that originallybelonged to the βA strand were separated from theirhydrogen-bonding partners of the βF strand. In addition, the inter-strand hydrogen bonds were also broken at Leu-186 and His-187 due to a main chain
distortion [Fig. 5(A–C)]. These conformationalchanges at the junction are expected to increase theﬂexibility of the relative positioning between the PDZ
tandem and the NZ-1 Fab. Glu-184 and His-187 werealso co-ordinated to a metal ion with a ﬁxed conformation in the co-crystal, but these side chains will
likely be solvent exposed and disordered under metalfree conditions. In contrast, we observed that theoverall fold of the target PDZ-N domain was hardly
affected by PA-insertion and Fab-binding. The 76 Cα
atoms of PDZ-N excluding the four junction residues,
Glu-184 to His-187, could be superposed onto to thoseof the wild type with a root-mean-square deviation(RMSD) of 0.697 Å.

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Figure 2. Complex formation of the PDZ tandem with the NZ-1 Fab through a PA-inserted PDZ-N domain. (A, B) Surface model of
PDZ tandem (181-PA12-184) in complex with the NZ-1 Fab in two different views. The inserted PA tag is shown in magenta. The
residues undergoing signiﬁcant structural changes compared with the wild type, as shown in Figure 5, are highlighted in yellow.
(C) Close-up view of the binding site. The PDZ-N domain and the inserted PA tag are shown as stick models with a transparent
surface. The solvent-accessible space between the rigidly folded part of the PDZ-N domain and the NZ-1 Fab is indicated with adouble-headed arrow. (D, E) Surface model of PDZ tandem (181-PA12-186) in complex with the NZ-1 Fab in two different views.
(F) Close-up view of the binding site. The models are colored as in (A, B, and C) while the PDZ domains are shown in pale colors.

Structural details of PDZ tandem (263-PA12-266)
in the Fab-bound form
The recognition mode of the PA tag was also conservedin the NZ-1 Fab-PDZ tandem (263-PA12-266) complex
where the Cα atoms of Gly-1 and Val-12 were separated by 12.1 Å [Fig. 4(C) and Supporting Information
Fig. S2(B)]. However, a slight difference was found in
the side chain conformation of Arg-120 on the NZ-1
heavy chain. It had been shown in the NZ-1 Fab-PA14
complex that this residue penetrated into the loop
formed by the PA tag and seemed to ﬁx the loop in theantigen-binding pocket where the guanidinium groupof Arg-120 interacted with the main chain carbonylgroups of Glu-8 and Asp-10 of PA14 [Fig. 4(A)]. In contrast, Arg-120 pointed to Arg-263 of PDZ-C and Gly-1
of PA12 in the NZ-1 Fab-PDZ tandem (263-PA12-266)
complex [Fig. 4(C)]. As in the above case of PDZ tandem (181-PA12-184), some junction residues of the βA
strand, such as Lys-266 and Met-267, were displacedby the PA-insertion [Fig. 6(A–C)]. In particular, Lys266 made no direct contacts with the NZ-1 Fab nor

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the remainder of PDZ-C [Figs. 3(C) and Supporting
Information Fig. S2(B)]. Both Lys-266 and Met-267
appeared to be highly mobile although they arelocated relatively close to one of the symmetry neighbors in the crystal packing.
Compared with the PDZ-N domain of PDZ tandem (181-PA12-184), the structure of the PA-inserted
PDZ-C domain deviated from that of the wild type toa greater extent. The 77 Cα atoms of PDZ-C excluding the PA tag were superposed on those of the wild
type with an RMSD of 0.980 Å. Unexpectedly, thelargest deviation was due to a conformational changeof the βA-βB loop, rather than one near the PAinsertion site. It had been found that the βA–βB loop
was relatively ﬂexible in the crystallographic analysisof the wild-type PDZ tandem.15 The conformation ofthis loop region is easily affected by lattice contacts
because it largely projects away from the domain.
Accordingly, the RMSD decreased to 0.696 Å when
the six residues belonging to the βA–βB loop were
excluded from the superposition.

Crystallizaion of PA-inserted target with NZ-1 FabFigure 3. Complex formation of the PDZ tandem with the NZ-1 Fab through a PA-inserted PDZ-C domain. (A, B) Surface model of
PDZ tandem (263-PA12-266) in complex with the NZ-1 Fab in two different views. The inserted PA tag is shown in magenta. The
residues undergoing signiﬁcant structural changes compared with the wild type, as shown in Figure 6, are highlighted in yellow.
(C) Close-up view of the binding site. The PDZ-C domain and the inserted PA tag are shown in stick models with a transparent
surface. The solvent-accessible space between the rigidly-folded part of the PDZ-C domain and the NZ-1 Fab is indicated with adouble-headed arrow. (D, E) Surface model of PDZ tandem (263-PA12-267) in complex with NZ-1 Fab in two different views.
(F) Close-up view of the binding site. The models are colored as in (A, B, and C) but the PDZ domains are shown in pale colors.

Optimization of PA-insertion sites to improvediffraction qualities of co-crystals
The above structural analysis does not support theconclusion that fold destabilization from insertion of
the PA tag caused the reduced resolution for the cocrystal structure. Apparently, the inserted PA tag did
not disrupt the fold topology of the PDZ domains ineither mutant. Another possibility was that the ﬂexibility between the PA-inserted PDZ domain and NZ-1
Fab increased the thermal vibration of the entire
complex in the crystal lattice and decreased the resolutions of the co-crystals. In both cases, the NZ-1 Fab
was tethered to the PDZ tandem mutants only
through the inserted PA tag, without direct contacts
with the remainder of the target PDZ tandem [Figs. 2(C) and 3(C)], while it formed lattice contacts withboth the NZ-1 Fab and the PDZ tandem from thesymmetry neighbors. Furthermore, some residues
located at the junction with the PA tag appeared tobe highly ﬂexible in both mutants, as mentioned
above. These observations suggested that the junction residues permit some extent of a swinging

Tamura et al.

motion of the NZ-1 Fab with respect to the PDZ
domain. Hence, we constructed two new mutants,
PDZ tandem (181-PA12-186) and (263-PA12-267), tomake more rigid complexes by deleting the respective
solvent-exposed residues at the junction sites.
The two modiﬁed mutants were stable and monodisperse after puriﬁcation, as shown by the elution
proﬁles of the size-exclusion chromatography [Supporting Information Fig. S1(E, F)]. We successfully
obtained co-crystals with the NZ-1 Fab for both ofthem. As hypothesized, the resolutions of the X-ray
diffraction were improved remarkably as compared tothose of the respective pre-modiﬁed mutants. The cocrystal structures with PDZ tandem (181-PA12-186)
and (263-PA12-267) were ﬁnally reﬁned to resolutions of 2.0 Å and 2.6 Å, respectively [Figs. 2(D–F)
and 3(D–F)]. In the complex with PDZ tandem(181-PA12-186), the 12.4 Å separation of the Cα
atoms in Gly-1 and Val-12 caused a ﬂip-out of
Leu-186 at the junction into the solvent, but theinter-strand hydrogen bonds with the βF strand
were maintained in the downstream of this residue

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Figure 4. Conformation of the PA tag in the antigen-binding pocket. (A) The PA14 peptide in complex with the NZ-1 Fab (PDB
code: 4YO0). The PA peptide is shown in magenta while the heavy and light chains of the NZ-1 Fab are colored with light orange
and light pink, respectively. Lys-76 and Arg-120 of the heavy chain are highlighted in blue where the hydrogen bonds formed by
the side chain of Arg-120 are indicated with dotted lines. Asp-75 of the heavy chain is also highlighted in red. (B) PDZ tandem(181-PA12-184) in complex with the NZ-1 Fab. The PDZ-N domain is shown in green where the residues showing signiﬁcant structural changes compared with the wild type are highlighted in yellow as shown in Figure 5. Metal ions presumed to be cadmiumions are shown with sphere models. (C) PDZ tandem (263-PA12-266) in complex with the NZ-1 Fab. The PDZ-C domain is shownin cyan where the residues showing signiﬁcant structural changes compared to the wild type are highlighted in yellow as shown in
Figure 6. (D) PDZ tandem (181-PA12-186) in complex with the NZ-1 Fab. (E) PDZ tandem (263-PA12-267) in complex with the NZ1 Fab. The models are colored as in previous ﬁgures but the PDZ-N and -C domains are shown in pale colors. The distances
between the Cα atoms of Gly-1 and Val-12 are indicated under the respective models.

[Figs. 4(D) and 5(D), and Supporting Information
Fig. S2(C)]. In the complex with the PDZ tandem(263-PA12-267), it appeared as if removing Lys-266
had no noticeable effect on the main chain conformation of the ﬂanking residues and the inter-strand

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hydrogen bonds [Fig. 6(A, D)]. The recognition modeof the PA tag as well as the distance between Gly-1
and Val-12 were essentially consistent with thosefrom other complexes [Figs. 4(E) and Supporting
Information Fig. S2(D)].

Crystallizaion of PA-inserted target with NZ-1 FabFigure 5. Structural change of the PDZ-N domain around the PA insertion site. (A) Superposition of the Cα traces. The wild-type
PDZ-N domain and the two PA-inserted mutants are displayed as varying shades of the same color scheme. The rigidly-foldedpart of the PDZ-N domain is shown in green where the residues undergoing signiﬁcant structural changes due to the PA insertion(Glu-184 to His-187) are highlighted in yellow. The two residues (Asn-182 and Gly-183) deleted to construct the PDZ tandem(181-PA12-184) are shown in dark magenta. The PA12 residues of PDZ tandem (181-PA12-184) and (181-PA12-186) are shown inbright magenta and light magenta, respectively. Close-up view of the PA insertion site of the wild type PDZ-N domain (B), PDZ
tandem (181-PA12-184) (C), and PDZ tandem (181-PA12-186) (D). Inter-strand hydrogen bonds are shown as orange dotted lines.

Although the removal of the junction residues
noticeably changed the orientation of the NZ-1 Fabwith respect to the PDZ tandem, the NZ-1 Fab again
made no direct contacts with the PDZ tandem otherthan at the inserted PA tag in both complexes(Figs. 2 and 3). It is, therefore, presumed that some
ﬂexibility still remained in the relative arrangement
between the NZ-1 Fab and the PDZ tandem. Again,
the PA-insertion and Fab-binding in these new
mutants has little effect on the fold of the target PDZ
domains. The 76 Cα atoms of PDZ-N in PDZ tandem(181-PA12-186) were superposed on their counterparts from the wild-type structure with an RMSD of
0.630 Å. The PDZ-C in PDZ tandem (263-PA12-267)

Tamura et al.

again showed a slightly larger structural change fromthat of the wild type, but the RMSD was reduced to
0.722 Å for 71 Cα atoms excluding the βA–βB loop
region. In contrast, the relative arrangement of PDZN and -C domains is ﬂexible and varies between theindividual models.

High-resolution structure of PDZ tandem(263-PA12-267) in the Fab-free form
In contrast to the pre-modiﬁed mutant, PDZ tandem(263-PA12-267) produced crystals in the Fab-freeform as well, which resulted in a structure determined to 1.9 Å resolution [Fig. 7(A)]. The electron
density of the inserted PA tag sequence was also

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Figure 6. Structural change of the PDZ-C domain around the PA insertion site. (A) Superposition of the Cα traces. The wild-type
PDZ-N domain and the two PA-inserted mutants are displayed as varying shades of the same color scheme. The rigidly foldedpart of the PDZ-C domain is shown in cyan where the residues undergoing signiﬁcant structural changes due to the PA insertion(Lys-266 and Met-267) are highlighted in yellow. The two residues (Asn-264 and Gly-265) deleted to construct the PDZ tandem(263-PA12-266) are shown in dark magenta. The PA12 residues of PDZ tandem (263-PA12-266) and (263-PA12-267) are shown inbright magenta and light magenta, respectively. Close-up view of the PA insertion site of the wild-type PDZ-C domain (B), PDZ
tandem (263-PA12-266) (C), and PDZ tandem (263-PA12-267) (D). Inter-strand hydrogen bonds are shown as orange dotted lines.

sufﬁciently clear to build the model. Of note, the PA
tag was partly incorporated into the β-hairpin structure of PDZ-C. Gly-1 and Val-2 participated in the βF
strand, and formed hydrogen bonds with Asp-10, Val11, and Val-12 included in the βA strand [Fig. 7(B)].
In addition, the Type II β-turn was constituted byMet-4, Pro-5, Gly-6, and Ala-7 in the typical conformation whereas the inter-strand hydrogen bonds
were disrupted at Ala-3, Glu-8, and Asp-9. The
extended β-hairpin protrudes out of PDZ-C to alength of about 16 Å, and it makes contacts with fourneighboring molecules in the crystal [Fig. 7(C)]. It
appears that the lattice contacts with the β-hairpin

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generate a rigidly packed crystal with a solvent content of 44.5%, which is much lower than that of theFab-free wild type crystal.
Comparison between the Fab-free and Fab-boundforms indicated that the structure of target PDZ-C
domain remarkably changed in Met-267 at the PA
tag junction as a result of Fab-binding. The βA–βB
loop of PDZ-C also showed a larger conformationalchange with a 6 Å shift at the Cα atom of Lys-279 in
the β-turn, which is presumably affected by latticecontacts. However, the main chain conformation wasmaintained between the Fab-free and Fab-boundforms with an RMSD of 0.823 Å for the 71 Cα atoms

Crystallizaion of PA-inserted target with NZ-1 FabFigure 7. Crystal structure of PDZ tandem (263-PA12-267) in the Fab-free form. (A) Ribbon model of the entire PDZ tandem. The
inserted PA tag and Met-267 are colored in magenta and yellow, respectively. The N- and C-terminal residues of the PA tag areincorporated into the β-strands of the target PDZ-C domain. (B) Stick model of the inserted PA tag. Inter-strand hydrogen bonds
are indicated with dotted lines. (C) Lattice contacts for PDZ tandem (263-PA12-267). PDZ tandem (263-PA12-267) is shown insurface model with the same coloring as in (A). The inserted PA tag makes direct contacts with four neighboring molecules(#1–#4) in the crystal lattice. (D) Conformational change of the inserted PA tag upon the Fab-binding. The Cα trace model in brightcolors represents the conformation in the Fab-bound form while the pale-colored model represents the Fab-free form. The NZ-1
Fab in the complex is shown as a surface model. Met-267 alone has undergone a conformational change in the target PDZ-C
domain. In contrast, Pro-5 and Gly-6 maintain a β-turn conformation after Fab-binding.

excluding the PA tag and the βA–βB loop region.
These observations indicate that the inserted PA tag
can adopt a conformation suitable for the interaction
with NZ-1 without affecting the folding of the target
PDZ-C domain [Fig. 7(D)].

Discussion
Application of antibody fragments has brought great
success for structural biology. In particular, crystallographic analyses of membrane proteins have been
accelerated dramatically by utilizing antibody fragments as crystallization chaperones.1,2,4 One beneﬁt
for crystallization from antibody fragments is
expanded solvent-exposed area in targets such as
membrane proteins. Antibody fragments are also useful for crystallizing other difﬁcult-to-crystallize proteins such as glycoproteins with conformational
ﬂexibilities.16 Most of the antibody fragment-assisted
crystallizations have been performed using an antibody that speciﬁcally recognizes the tertiary structureof the target protein. Our study further raised the

Tamura et al.

possibility that an anti-tag antibody can be applied tothe same kind of crystallization strategy under thecondition that the tag sequence can be inserted into
the target protein without disrupting its tertiary structure. In the present work, we selected the β-hairpinsin the PDZ tandem fragment of a bacterial S2P homolog as the insertion sites because the Fab-bound PA
tag is known to adopt a loop-like structure with aβ-turn between Pro-5 and Gly-6. Although the size of
the PA tag is not negligible compared with the total
size of the individual 80-residue PDZ domains, thePA-insertion did not disrupt the fold topology of thetarget PDZ domains for any of the mutants constructed in the present work (Supporting Information
Fig. S1). Furthermore, our results have shown thatthe inserted PA tag can serve as a scaffold to generatea ready-to-crystallize complex between the NZ-1 Faband the target PDZ tandem. In fact, we obtained cocrystals with the NZ-1 Fab for all of the PA-inserted
mutants, even in cases where crystallization was not
successful for the Fab-free form.

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In addition, our results strongly indicated that
optimization of the insertion site should be an important process for obtaining a co-crystal with a higher
diffraction quality. We attempted to insert the PA tag
into the two different sites and initially obtained cocrystals with moderate diffraction qualities for bothcases. Based on our inspection of the co-crystal structures, we hypothesized that the conformational ﬂexibilities between the NZ-1 Fab and the target PDZ
domains lowered the diffraction qualities. The NZ-1
Fab did not form any speciﬁc interactions with thetarget PDZ domains, but was tethered to them only
through the inserted PA tag within the complex[Figs. 2(C, F) and 3(C, F)]. Hence, the solventaccessible space present between the NZ-1 Fab andthe rigidly folded part of the PDZ domain is thought
to increase thermal vibration throughout the entire
complex. To test the hypothesis, we modiﬁed both of
the two mutants by eliminating the ﬂexible residues
present at the insertion sites. Ultimately, the two
modiﬁed mutants produced the co-crystals withhigher diffraction qualities than those of the respective pre-modiﬁed mutants. The averaged temperaturefactors in the co-crystals signiﬁcantly decreased after
the modiﬁcation of the insertion sites (Table II),
although it is not a direct measurement of decreased
conformational ﬂexibilities in the complexes. Remarkably, the diffraction qualities were also higher thanthat of the Fab-free wild-type crystal. These results
support the case that the NZ-1 Fab plays a beneﬁcialrole in the crystallization and improved diffraction
quality of the PDZ tandem mediated through theinserted PA tag.
The structure determination of PDZ tandem(263-PA12-267) has demonstrated that the inserted
PA has potential to form a β-hairpin structure. The
formation of the β-hairpin structure of the PA tag in
the Fab-free PDZ tandem (263-PA12-267) crystal isprobably not only affected by the lattice contact but
also dependent on the sequence context. Nevertheless,
it is highly possible that β-hairpins are generally competent as PA-insertion sites. As the Pro-Gly sequence
generally has a higher β-turn propensity,17,18 it is presumed that Pro-5 and Gly-6 at the middle of the PA
tag sequence are predisposed to form a turn at theinsertion site. When incorporated as a β-hairpin, thePA tag is expected to dock to the NZ-1 Fab with anintact β-turn conformation between Pro-5 and Gly-6.
Furthermore, it is noteworthy that other than for Met267 the main chain conformations of the residues
ﬂanking the PA tag are almost identical in both theFab-free and Fab-bound PDZ tandem (263-PA12-267)[Fig. 7(D)]. It seems that the inter-strand hydrogenbonds maintained the folding of the target PDZ
domain during the Fab-binding, which also suggeststhat the β-hairpins are suitable sites for PA insertion.
Although we here inserted the PA tag into a protein for which the 3D structure was initially

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available, it is presumed that the same cocrystallization strategy can be applied to proteins
whose 3D structures have not yet been determined
experimentally. Accumulation of structural data inProtein Data Bank (PDB) and development of structure prediction algorithms19,20 should enable us topredict the positions of loop regions that become candidates for PA-insertion sites in proteins without an
initial mid- to low-resolution structure model. For
instance, glycoproteins secreted to extracellularspace or ectodomain fragments of membrane proteins should be ideal targets for the PA insertion as
they are abundant in the β-hairpin structures.
Although multi-pass membrane proteins are generally α-helical in the transmembrane regions, the loop
regions connecting the α-helices can be candidates
for the insertion sites. Based on our structural analysis, an additional criterion can be proposed to selectcandidate insertion sites. It has been shown in thecomplex structures that docking to the NZ-1 Fabseparates Gly-1 and Val-12 of the PA tag by at least
12 Å (Fig. 4). Therefore, PA tag insertion sites that
conform to this distance constraint can be selectedfrom a predicted structural model. In fact, some residues belonging to the βA strand were deleted alongwith the loop residues for PA insertion in the two
modiﬁed mutants of the PDZ tandem. The additionaldeletions extended the end-to-end distance of the PA
tag in the host proteins and thereby minimized thestructural distortion upon the Fab-binding in both
two cases. We expect some difﬁculty in ﬁnding an
optimal insertion site in the case of de novo structure determination. Nevertheless, once initial cocrystal structures can be determined it should bepossible in principle to improve the diffraction qualities of co-crystals from low-resolution starting points
by optimizing the insertion sites, as we demonstrated in this work.
In addition to the merit for protein crystallography, it is also highly probable that complex formation
with an antibody fragment through an inserted epitope will be advantageous for structural determination via cryo-EM. In recent years, cryo-EM analysis
has undergone dramatic development and has produced a growing number of atomic models for
difﬁcult-to-crystallize targets.21–23 However, it is still
difﬁcult to resolve high-resolution cryo-EM structures
for targets with smaller particle sizes and lower symmetries. Several characteristics of our approachshould have beneﬁt toward these technical challenges
in cryo-EM analysis. An antibody fragment bound to
the target should increase the size of the particle andserve to identify the orientation. While the conformational ﬂexibility between the target and the boundantibody is expected to disturb the averaging procedure, it could be addressed by optimizing the insertion site based on the preliminary structural analysis
as discussed above.

Crystallizaion of PA-inserted target with NZ-1 FabConclusion
The present work demonstrates that the NZ-1-PA system has potential to be applied to antibody fragmentassisted crystallization provided that the insertion
sites of the PA tag are properly selected and optimized. In particular, proteins possessing β-hairpins
are promising candidates for PA-insertion targets. The
next issue to be addressed is whether or not our strategy can be applied to structure determination of more
difﬁcult targets such as α-helical integral membraneproteins and modular glycoproteins with larger molecular sizes.

Materials and Methods
Puriﬁcation of the PA-inserted PDZ tandem
mutants in the Fab-free and Fab-bound forms
The PDZ tandem fragment (Residues 115–292) was
produced as an N-terminal GST (glutathione S transferase) -fusion protein, in which a TEV protease recognition site was incorporated between the GST and
PDZ tandem sequences, as reported previously.15
Each PA-inserted construct was prepared by amplifying the whole plasmid DNA by PCR using a primer
containing the DNA sequence of the PA tag. Escherichia coli BL21(DE3) cells transformed with theexpression plasmid were grown at 37 C to an OD600
of 0.7 in a medium containing 10 g of bactotryptone,
5 g of yeast extract and 10 g of NaCl per liter supplemented with 50 μg/mL ampicillin, followed by induction of overexpression with 0.1 mM isopropyl
β-D-thiogalactopyranoside and incubation at 25 C for
additional 12 h. Cells were harvested by centrifugation and lysed by sonication in Tris buffered saline(TBS) composed of 10 mM Tris-Cl (pH 7.4) and
150 mM NaCl. The soluble fraction of the cell lysate
was mixed with Glutathione-Sepharose 4B resin(GE Healthcare UK Ltd, Buckinghamshire, England)
and incubated at 4 C for 1 h. After washing out theunbound fraction with TBS, the PDZ tandem wascleaved off from the GST portion through on-column
digestion with TEV protease at 20 C for 12 h. The
released PDZ tandem fragment was further puriﬁedusing cation-exchange chromatography HiTrap SP
HP (GE Healthcare UK Ltd) and size-exclusion chromatography Superdex 200 Increase 10/300 GL(GE Healthcare UK Ltd). Gel Filtration Standard(Bio-Rad) was used as the molecular weight marker
in size-exclusion chromatography. The puriﬁed PDZ
tandem was mixed with the NZ-1 Fab at a molar
ratio of 2:1 and incubated on ice for 30 min. NZ-1 was
obtained from the Antibody Bank (http://www.medtohoku-antibody.com/topics/antibody.htm) at Tohoku
University (Miyagi, Japan). Brieﬂy, NZ-1 was produced in Hybridoma-SFM (Thermo Fisher ScientiﬁcInc., Waltham, MA, USA), and was puriﬁed using
Protein G (GE Healthcare UK Ltd). The NZ-1 Fab
was prepared by papain digestion according to the

Tamura et al.

protocol as described previously.11 The mixture of
PDZ tandem and NZ-1 Fab was applied to sizeexclusion chromatography again to separate the PDZ
tandem fragments into the Fab-bound and Fab-freeforms. The ﬁnal protein samples were concentrated
by ultraﬁltration.

Crystallization and data collection
The initial crystallization screens were conductedusing Index™ (Hampton Research) crystallization
reagents. About 0.2 μL of the protein solutions andreagents, respectively, were dispensed into 96-wellplates using a Gryphon robotic crystallization system(Art Robbins Instruments) and equilibrated against
60 μL of the reservoir by the sitting-drop vapordiffusion method. The crystals subjected to X-ray
crystallographic analysis were generated from crystallization buffers as follows. The Fab-bound PDZ
tandem (181-PA12-184) was crystallized in buffer
containing 5 mM CoCl2, 5 mM NiCl2, 5 mM CdCl2,
5 mM MgCl2, 100 mM HEPES-Na (pH 7.5), and 12%(wt./vol.) polyethylene glycol (PEG) 3350. The crystalsof the Fab-bound PDZ tandem (263-PA12-266) wereobtained in buffer containing 200 mM ammonium citrate (pH 7.0), and 20% (wt./vol.) PEG 3350. For Fabbound PDZ tandem (181-PA12-186), the crystallization buffer contained 200 mM sodium citrate, and12–15% (wt./vol.) PEG 3350. For PDZ tandem(263-PA12-267), the crystal of the Fab-bound form
was obtained from buffer containing 200 mM ammonium citrate (pH 7.0), and 15–20% (wt./vol.) PEG
3350 while the Fab-free form was crystallized inbuffer containing 200 mM Bis-Tris-Cl (pH 6.5), and
23–25% (wt./vol.) PEG 3350. Cryoprotectant was
prepared by mixing the crystallization buffer andethylene glycol with a volume ratio of 4:1. However,the crystals of the Fab-bound PDZ tandem(181-PA12-184) were labile in the cryoprotectant
prepared according to this protocol. Therefore, we
prepared cryoprotectant containing the same concentration of precipitant as the crystallization buffer
to prevent the crystals from dissolving. All of thecrystals were quickly soaked in the respective cryoprotectant and frozen in liquid nitrogen. X-ray diffraction data were collected with a photon counting
pixel array detector Eiger X4M (Dectris) at Photon
Factory (PF) BL-1A (Tsukuba, Japan) or with PILATUS3 S 6M (Dectris) at PF BL-17A. Data were
processed and scaled with XDS24 and aimless.25
Intensities were converted to structure factors with
the CCP4 programs where 5% of the unique reﬂections were randomly selected as a test set.26,27 Data
collection statistics are summarized in Table I.

Crystallographic analysis
For each crystal, initial phases were determined by
the molecular replacement (MR) method by using
Molrep28 in CCP4. The atomic coordinates of the

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833

Table I. Data Collection Statistics
Construct
Form
Space group
Cell dimensions
a, b, c (Å)α, β, γ ( )No. of monomers
or complexes/a.s.u.
X-ray source
Wavelength (Å)
Resolution limits (Å)
No. of unique reﬂection
Completeness (%)
Redundancy
I/σ(I)
RmergeaCC (1/2)

PDZ tandem(181-PA12-184)

PDZ tandem(181-PA12-186)

PDZ tandem(263-PA12-266)

PDZ tandem(263-PA12-267)

PDZ tandem(263-PA12-267)

Fab-boundP212121Fab-boundP212121Fab-boundI422

Fab-boundP4212

Fab-free
P21212162.67, 83.04,
188.8890, 90, 90
149.94, 84.29,
185.9190, 90, 90
1171.93, 171.79,
118.0290, 90, 90
1141.65, 141.68,
78.62
90, 90, 90
149.23, 54.80,
68.32
90, 90, 90
1PF/BL-1A
1.100048.35–3.20(3.42–3.20)
16,978 (3,004)
100.0 (100.0)
6.6 (6.8)
12.6 (1.8)
0.124 (1.170)
0.932 (0.916)

PF/BL-17A
0.980048.23–2.00(2.05–2.00)
54,069 (3,874)
100.0 (100.0)
6.7 (6.7)
16.1 (2.3)
0.056 (0.874)
0.940 (0.873)

PF/BL-1A
1.100048.64–4.00(4.47–4.00)
7,742 (2,144)
99.9 (100.0)
13.5 (12.5)
12.2 (3.9)
0.186 (0.744)
0.996 (0.911)

PF/BL-1A
1.100050.09–2.60(2.72–2.60)
25,220 (3,024)
100.0 (100.0)
17.9 (18.5)
12.5 (4.1)
0.183 (0.808)
0.993 (0.924)

PF/BL-1A
1.100047.25–1.90(1.94–1.90)
15,147 (948)100.0 (100.0)
13.4 (13.2)
10.6 (2.1)
0.176 (2.746)
0.997 (0.790)

Values in parentheses are for highest-resolution shell.
Rmerge = ΣhΣi|Ii(h)− < I(h) > |/ΣhΣiI(h), where Ii (h) is the ith measurement.

aA. aeolicus PDZ tandem15 (PDB code: 3WKL) andNZ-1 Fab with the bound PA14 peptide11 (PDB code:
4YO0) were used as search models. As the PDZ tandem arrangement is conformationally ﬂexible, themodel was separated into two portions, the PDZ-Nand -C domains. Similarly, the Fab model was also
separated into the Fv and constant regions.

Accordingly, the four partial structures were used as
the search models in the calculation of MR phasing.
After assignment of the respective models, manualﬁtting was performed with COOT29 and the updated
models were reﬁned with Refmac530 iteratively. For
the Fab-bound PDZ tandem (181-PA12-186) and theFab-free PDZ tandem (263-PA12-267), the initial

Table II. Reﬁnement Statistics
Construct
Form
Resolution
limits (Å)
RworkaRfreeaNo. of atoms
PDZ tandem
PDZ-N
PDZ-C
NZ-1 Fab
Solvent
Average B-factor (Å2)
PDZ tandem
PDZ-N
PDZ-C
NZ-1 Fab
Solvent
RMSD from ideality
Bond length (Å)
Bond angle ( )Ramachandran plot
Favored (%)
Outlier (%)
PDB code

PDZ tandem(181-PA12-184)

PDZ tandem(181-PA12-186)

PDZ tandem(263-PA12-266)

PDZ tandem(263-PA12-267)

PDZ tandem(263-PA12-267)

Fab-bound48.35–3.20(3.28–3.20)
0.278 (0.442)
0.313 (0.457)
3,928
745
745
0
3,183
0
115.31121.57121.57113.85-

Fab-bound48.23–2.00(2.05–2.00)
0.213 (0.312)
0.260 (0.314)
5,046
1,470
787
683
3,325
251
47.64
50.16
46.53
54.35
46.22
51.55

Fab-bound48.64–4.00(4.10–4.00)
0.216 (0.304)
0.273 (0.347)
4,724
1,439
714
725
3,285
0
132.48146.55160.94132.37126.32-Fab-bound50.09–2.60(2.67–2.60)
0.199 (0.294)
0.251 (0.403)
5,012
1,480
755
725
3,350
182
42.22
55.99
63.81
47.85
36.46
36.09

Fab-free42.75–1.90(1.95–1.90)
0.200 (0.286)
0.254 (0.392)
1,581
1,482
751
731
99
32.32
31.92
31.34
32.52
38.25

0.002
0.73

0.004
1.11

0.008
0.82

0.004
1.06

0.008
1.67

92.45
0.60
6AL1

98.36
0.16
6ICC

92.76
0.82
6ICF

96.36
0
6AL0

98.90
0
6AKQ

Values in parentheses are for highest-resolution shell.
Rwork is the crystallographic R-factor (Rcryst) for the working set used for the reﬁnement. Rcryst = Σh||Fobs(h)|−|Fcalc(h)||/Σh| Fobs(h)|, where Fobs(h) and Fcalc(h) are the observed and calculated structure factors, respectively.
b
Rfree is Rcryst calculated for the test set consisting of 5% of reﬂections excluded from the reﬁnement.
a834

PROTEINSCIENCE.ORG

Crystallizaion of PA-inserted target with NZ-1 Fabmodels were improved by automatic model building
with ARP/wARP31 prior to manual ﬁtting. For the
Fab-bound PDZ tandem (181-PA12-184), the electron
densities indicated the presence of metal ions boundto the protein surface. These metal ions were presumed to be cadmium ions included in the crystallization buffer. We displayed models of two ions together
with the co-ordinated amino acid residues in Figure 4(B) and Figure S2(A). However, we deleted the ionsfrom the ﬁnal model deposited in PDB because theentities of the electron densities could not be unambiguously assigned as explained in the Results
section.
Stereochemical parameters of the ﬁnal models
were assessed with MolProbity.32 Reﬁnement statistics are summarized and PDB accession codes arelisted in Table II. Structural superposition and
RMSD calculation were performed by the pair-wisealignment protocol using LSQKAB.33 Figures for protein structures were prepared with PyMOL (ThePyMOL Molecular Graphics System, Version 1.8
Schrödinger, LLC.).

Acknowledgments
We are grateful to the beamline staff of Photon Factory (Tsukuba, Japan) for providing data collection
facilities and support. We thank Makiko Neyazaki fortechnical support, Samuel Thomson for editing themanuscript, and Prof Junichi Takagi for useful discussion. This research is partially supported by theJapan Society for the Promotion of Science (JSPS)
KAKENHI Grant nos. JP26291016 and JP17K19206(to T.N.), by the Platform Project for Supporting in
Drug Discovery and Life Science Research (Platformfor Drug Discovery, Informatics, and Structural Life
Science) from Japan Agency for Medical Researchand Development (AMED) under Grant no.
JP16am0101020 (to T.N.), and by the Platform Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from
AMED under Grant no. JP17am0101078 (to Y.K.).

Competing interests
The corresponding author declares no ﬁnancial andnon-ﬁnancial competing interests on behalf of all
authors.

References
1. Ostermeier C, Iwata S, Ludwig B, Michel H (1995) Fv
fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase. Nat Struct Biol 2:
842–846.
2. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R(2001) Chemistry of ion coordination and hydration
revealed by a K+ channel-Fab complex at 2.0 a resolution. Nature 414:43–48.

Tamura et al.

3. Hunte C, Michel H (2002) Crystallisation of membraneproteins mediated by antibody fragments. Curr Opin
Struct Biol 12:503–508.
4. Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS,
Thian FS, Edwards PC, Burghammer M, Ratnala VR,
Sanishvili R, Fischetti RF, Schertler GF, Weis WI,
Kobilka BK (2007) Crystal structure of the human beta2
adrenergic G-protein-coupled receptor. Nature 450:
383–387.
5. Day PW, Rasmussen SG, Parnot C, Fung JJ, Masood A,
Kobilka TS, Yao XJ, Choi HJ, Weis WI, Rohrer DK,
Kobilka BK (2007) A monoclonal antibody for G proteincoupled receptor crystallography. Nat Methods 4:
927–929.
6. Hino T, Iwata S, Murata T (2013) Generation of functional antibodies for mammalian membrane protein
crystallography. Curr Opin Struct Biol 23:563–568.
7. Bradbury AR, Sidhu S, Dubel S, McCafferty J (2011)
Beyond natural antibodies: the power of in vitro display
technologies. Nat Biotech 29:245–254.
8. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H,
Mishima K, Osawa M (2006) Inhibition of tumor cellinduced platelet aggregation using a novel anti-podoplaninantibody reacting with its platelet-aggregation-stimulating
domain. Biochem Biophys Res Commun 349:1301–1307.
9. Ogasawara S, Kaneko MK, Price JE, Kato Y (2008)
Characterization of anti-podoplanin monoclonal antibodies: critical epitopes for neutralizing the interaction
between podoplanin and CLEC-2. Hybridoma (Larchmt)
27:259–267.
10. Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y,
Takagi J (2014) PA tag: a versatile protein tagging system using a super high afﬁnity antibody against a dodecapeptide derived from human podoplanin. Prot Expr
Purif 95:240–247.
11. Fujii Y, Matsunaga Y, Arimori T, Kitago Y,
Ogasawara S, Kaneko MK, Kato Y, Takagi J (2016) Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational
state. J Cell Sci 129:1512–1522.
12. Wang H, Han W, Takagi J, Cong Y (2018) Yeast innersubunit PA-NZ-1 labeling strategy for accurate subunit
identiﬁcation in a macromolecular complex throughcryo-EM analysis. J Mol Biol 430:1417–1425.
13. Brown ZP, Arimori T, Iwasaki K, Takagi J (2018) Development of a new protein labeling system to map subunits and domains of macromolecular complexes forelectron microscopy. J Struct Biol 201:247–251.
14. Deckert G, Warren PV, Gaasterland T, Young WG,
Lenox AL, Graham DE, Overbeek R, Snead MA,
Keller M, Aujay M, Huber R, Feldman RA, Short JM,
Olsen GJ, Swanson RV (1998) The complete genome ofthe hyperthermophilic bacterium Aquifex aeolicus.
Nature 392:353–358.
15. Hizukuri Y, Oda T, Tabata S, Tamura-Kawakami K,
Oi R, Sato M, Takagi J, Akiyama Y, Nogi T (2014) A
structure-based model of substrate discrimination by anoncanonical PDZ tandem in the intramembranecleaving protease RseP. Structure 22:326–336.
16. Grifﬁn L, Lawson A (2011) Antibody fragments as tools
in crystallography. Clin Exp Immunol 165:285–291.
17. Hutchinson EG, Thornton JM (1994) A revised set of
potentials for beta-turn formation in proteins. Protein
Sci 3:2207–2216.
18. Hsu HJ, Chang HJ, Peng HP, Huang SS, Lin MY,
Yang AS (2006) Assessing computational amino acidbeta-turn propensities
with aphage-displayed

PROTEIN SCIENCE | VOL 28:823–836

835

combinatorial library and directed evolution. Structure
14:1499–1510.
19. Petersen B, Lundegaard C, Petersen TN (2010) NetTurnP--neural network prediction of beta-turns by useof evolutionary information and predicted protein
sequence features. PLoS One 5:e15079.
20. Zheng C, Kurgan L (2008) Prediction of beta-turns atover 80% accuracy based on an ensemble of predictedsecondary structures and multiple alignments. BMC
Bioinformatics 9:430.
21. Cheng Y (2018) Single-particle cryo-EM – How did it get
here and where will it go. Science 361:876–880.
22. Merk A, Bartesaghi A, Banerjee S, Falconieri V, Rao P,
Davis MI, Pragani R, Boxer MB, Earl LA, Milne JLS,
Subramaniam S (2016) Breaking cryo-EM resolution
barriers to facilitate drug discovery. Cell 165:
1698–1707.
23. Kuhlbrandt W (2014) Biochemistry. The resolution revolution. Science 343:1443–1444.
24. Kabsch W (2010) Xds. Acta Crystallogr D66:125–132.
25. Evans PR, Murshudov GN (2013) How good are my data
and what is the resolution? Acta Cryst D69:1204–1214.
26. Collaborative Computational Project Number 4 (1994)
The CCP4 suite: programs for protein crystallography.
Acta Cryst D50:760–763.

836

PROTEINSCIENCE.ORG

27. Winn MD, Ballard CC, Cowtan KD, Dodson EJ,
Emsley P, Evans PR, Keegan RM, Krissinel EB,
Leslie AG, McCoy A, McNicholas SJ, Murshudov GN,
Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A,
Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Cryst D67:235–242.
28. Vagin A, Teplyakov A (1997) MOLREP: an automatedprogram for molecular replacement. J Appl Cryst 30:
1022–1025.
29. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Cryst D66:
486–501.
30. Murshudov GN, Vagin AA, Dodson EJ (1997) Reﬁnement of macromolecular structures by the maximumlikelihood method. Acta Cryst D53:240–255.
31. Perrakis A, Harkiolaki M, Wilson KS, Lamzin VS (2001)
ARP/wARP and molecular replacement. Acta Cryst D57:
1445–1450.
32. Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA,
Immormino
RM,
KapralGJ,
Murray
LW,
Richardson JS, Richardson DC (2010) MolProbity: allatom structure validation for macromolecular crystallography. Acta Cryst D66:12–21.
33. Kabsch W (1976) A solution for the best rotation torelate two sets of vectors. Acta Cryst A32:922–923.

Crystallizaion of PA-inserted target with NZ-1 Fab