TEC H NICAL RE P ORTS
Precise mapping of subunits in multiprotein complexes by
a versatile electron microscopy label
Q1
Dirk Flemming1, Karsten Thierbach1, Philipp Stelter1, Bettina Böttcher2 & Ed Hurt1

Positional knowledge of subunits within multiprotein
assemblies is crucial for understanding their function.
The topological analysis of protein complexes by electron
microscopy has undergone impressive development, but
analysis of the exact positioning of single subunits has lagged
behind. Here, we have developed a clonable ~80-residue
tag that, upon attachment to a target protein, can recruit a
structurally prominent electron microscopy label in vitro.
This tag is readily visible on single particles and becomes
exceptionally distinct after image processing and classification.
Thus, our method is applicable for the exact topological
mapping of subunits in macromolecular complexes.
Structure determination of macromolecular assemblies by singleparticle electron microscopy analysis has seen impressive progress
in recent years. As such, the precise positional mapping of subunits
within a multisubunit complex at intermediate resolution becomes
even more imperative to allow interpretation of the macromolecular architecture1,2. Several methods for marking proteins in complex
assemblies and subsequent visualization by electron microscopy have
been reported, including antibody and gold labeling, attachment of
green fluorescent protein (GFP) or attachment of an actin-binding
protein domain to the target protein with subsequent recruitment of
an electron microscopy–visible actin filament3–7. However, although
each of these methods has certain advantages, each also has drawbacks, such as low labeling efficiency and/or a high flexibility of the
bound label, that impede the determination of the precise topological
position of a given subunit within the protein complex. As a conseq­
uence, large datasets often are needed to determine the location of the
label in sophisticated image-processing approaches.
Here, we have developed a new tool for topological mapping of individual subunits within a multimeric protein assembly by exploiting a
structurally prominent label that is easily identifiable by negative-stain
electron microscopy. We discovered this prominent structure when
studying the binding of yeast dynein light chain (Dyn2) to a dynein
light chain–interacting domain (DID) in the nucleoporin Nup159
(ref. 8). Typically, a dynein light chain dimer can accommodate two
~12-mer peptides with the Gln-Thr (‘QT’) recognition motif on the two
opposing binding pockets of the homodimer9. In vitro reconstitution of
the Dyn2–DID complex from recombinant proteins revealed a prominent rod-like structure in which five Dyn2 homodimers were aligned
between two extended DID strands like pearls on a string8. Thus, we
sought to take advantage of this tool as a versatile electron microscopy
tag to mark individual proteins in multisubunit complexes.
RESULTS
Design and structure of the DID–Dyn2 electron microscopy label
To develop the electron microscopy labeling tool, we used two different DID strands, one derived from Pac11 (DID1; see Online Methods)
and the other from Nup159 (DID2) (Fig. 1a). Both contained six predicted, consecutive QT consensus motifs so that these two strands
could sandwich six Dyn2 homodimers when arranged in an opposing
parallel manner (Fig. 1b). To reconstitute in vitro the predicted DID1–
Dyn2–DID2 complex with recombinant proteins, we first immobilized
Protein A (ProtA)-TEV-DID1 on IgG-Sepharose before adding Dyn2
plus Flag-DID2 to the beads. A noteworthy aspect of the ­applicability
of our labeling method is that the needed recombinant Dyn2 and FlagDID2 can be easily purified from Escherichia coli in high yield and
can be stably stored in small aliquots at −80 °C. After assembly of the
DID strands with Dyn2, we eluted it with the TEV protease before we
reisolated the complex on an anti-Flag Sepharose column followed by
Flag peptide elution (see also below). Notably, this assembled complex
was stable over a wide range of salt ­conditions (0 mM to 1 M NaCl; data
not shown). When we analyzed this DID1–Dyn2–DID2 complex (~130
kDa) by negative-stain electron microscopy, we found on the micrographs a readily observable ~25-nm-long rod-like filament that, upon
alignment and classification, showed six distinct globular domains in a
row (Fig. 1c). These data suggest that six Dyn2 homodimers were sandwiched between the oppositely aligned DID1 and Flag-DID2 strands.
Strategy and labeling of the Nup84 complex by the DID–Dyn2 rod
To test whether this prominent DID–Dyn2 structure can be attached
to a subunit of a multimeric complex and subsequently visualized
by electron microscopy, we sought to use the Nup84 complex, an
essential nuclear pore complex module that has been well studied
by electron microscopy in the past10,11. Previously, the position of
the nucleoporin Seh1 within the Nup84 complex was determined by
GFP labeling of this subunit at the C terminus followed by electron
1Biochemie-Zentrum
der Universität Heidelberg, Heidelberg, Germany. 2The School of Biological Sciences, University of Edinburgh, Scotland, UK. Correspondence
should be addressed to E.H. ([email protected]).
Received 5 January; accepted 19 March; published online XX XXXX 2010; doi:10.1038/nsmb.XX
nature structural & molecular biology advance online publication
TEC H NICAL RE P ORTS
Figure 1 Design of the DID–Dyn2 electron microscopy label. (a) Residue
sequence of the two DID strands, DID1 (derived from Pac11) and Flagtagged DID2 (derived from Nup159). The six ‘QT’ recognition motifs
(12-mer peptides) in each strand are highlighted in green, the Flag motif
at the N terminus of DID2 in brown. (b) Cartoon showing the assembled
electron microscopy label consisting of six Dyn2 homodimers (blue)
sandwiched between the two DID strains (green), DID1 and Flag-DID2.
(c) Electron microscopy micrograph overview of the affinity-purified
DID–Dyn2 complex (left; scale bar, 100 nm) and two representative class
averages showing six globular masses in a row (right; scale bar, 10 nm).
a
DID1
Flag-DID2
b
Dyn2-Dyn2
DID1
microscopy11. A GFP density at one arm of the Nup84 complex only
became evident in class-average images and appeared to be fuzzy,
possibly due to flexibility of the attached GFP11. Additionally, the
position of Nup120 within this assembly has been determined indirectly by docking of the Nup120 crystal structure into the electron
microscopy envelope12.
To test our labeling strategy, we fused DID1 by recombinant techniques to either Seh1 or Nup120 (see Fig. 2a). Subsequently, we
expressed the pentameric Nup84 complexes (Nup120–Nup145C–
Nup85–Sec13–DID1-Seh1 or DID1-Nup120–Nup145C–Nup85–
Sec13–Seh1) in E. coli and affinity-purified them via GST-labeled
Nup145C. The attachment of the short DID1 to either Seh1 or Nup120
did not interfere with the assembly of the Nup84 complex. Indeed, it
showed the same biochemical composition and stoichiometry as the
unmodified Nup84 complex, with the exception that the ­molecular
weight of DID1-Seh1 or DID1-Nup120 was shifted according to the
attached tag (Fig. 2b and Supplementary Fig. 1). Moreover, the
overall Y-shaped structure of the DID1-modified Nup84 ­ complex
was indistinguishable from the untagged complexes (Fig. 2c; see also
Fig. 3), suggesting that the DID1 sequence could be flexible and/or
unfolded (see also ref. 8). Finally, the Y-shaped Nup84 complex
­carrying either DID1-Seh1 or DID1-Nup120 showed the same
Glutathione Agarose
a
DID
b
200
Flag
GST
DID
Nup120
Nup85
Nup145C
Dyn2 dimers
1
Seh1
1. GSH binding
2. Incubation with
Dyn2 + Flag-DID2
Glutathione Agarose
kDa
2
100
50
c
Nup84 complex–DID1-Seh1
without Dyn2
Overview
Overview
Gallery
Gallery
5. Flag peptide elution
Dyn
2–D
Protein complex
ID la
bel
s­ tructural features as those described for the heptameric complex,
except that one arm of the triskelion was shorter due to the absence
of the elongated Nup84–Nup133 module10,11.
For the actual labeling procedure, we immobilized the Nup84
complex carrying DID1-Seh1 (or DID1-Nup120) on GSH beads via
GST-TEV-Nup145C and incubated it with Dyn2 plus the ­ second
Flag-DID2 strand to develop the structural electron microscopy
marker on the assembled Nup84 complex. For this step, we added
an excess of purified Dyn2 homodimer and Flag-DID2 to the beads
and incubated them for 1 h or longer at 4 °C (for scheme, see Fig. 2a).
After the recruitment of six Dyn2 homodimers to DID1-Seh1 (or
DID1-Nup120) and binding of the second Flag-DID2 strand, we
released the pentameric Nup84 complex (now carrying the DID–
Dyn2 label) by TEV cleavage, generally for
45 min at 16 °C or for 16 h at 4 °C. Finally, we
­further enriched the Nup84 complex with the
structural DID–Dyn2 label on an anti-Flag
Nup120
Sepharose column before elution with Flag
Nup85
Nup145C
peptides (Fig. 2a). In total, the building up
DID1-Seh1
of the label, starting from the ­immobilized
Nup84 complex–DID1-Seh1
Dyn2 + Flag-DID2
α-FLAG-Sepharose
3. TEV cleavage
4. Anti-Flag binding
1
2
3
4
5
6
Flag
Dyn2
10
DID1–Dyn2–Flag-DID2
1
2
3
4
5
6
Sec13
Flag-DID2
Sec13
TEV
DID2
c
Figure 2 Labeling of the Seh1 subunit within
the Nup84 complex with the DID–Dyn2 electron
microscopy marker. (a) Schematic flow chart
of the labeling and purification procedure
to reconstitute the DID–Dyn2 marker on the
Nup84 complex (see text for details).
(b) SDS-PAGE and Coomassie blue staining of
the purified Nup84 complex (left, unlabeled
complex) and of the Nup84 complex carrying
DID1-Seh1 and labeled with Dyn2 and
Flag-DID2. This labeled complex was finally
affinity-purified on anti-Flag Sepharose followed
by Flag peptide elution. The weaker Coomassie
staining of the Flag-DID2 band (12.2-kDa
calculated molecular weight) is explained by
its abnormal running behavior on the SDSPAGE gel. (c) Micrograph overview and gallery
of single particles of the purified DID1-Seh1
carrying Nup84 complex without (left) and
with reconstituted Dyn2-Flag-DID2 label (right).
Scale bars, 50 nm (overview) and 10 nm
(single particles).
advance online publication nature structural & molecular biology
TEC H NICAL RE P ORTS
Figure 3 Aligned class averages of unlabeled and DID–Dyn2–labeled
Nup84 complex. (a) Representative electron microscopy classes of the
unmodified Nup84 complex (Nup120–Nup145C–Nup85–Seh1–Sec13;
left) and the Nup84 complex carrying DID1-Seh1 but without addition
of Dyn2 and Flag-DID2 (right). (b,c) Representative electron microscopy
classes of DID1-Seh1 (b) and DID1-Nup120 (c) carrying Nup84 complex
with bond Dyn2–Flag-DID2 label. The label (arrowhead) and the three
different arms (1, 2 and 3) of the Nup84 complex are indicated. Scale,
10 nm. (d) Model of the Nup84 complex carrying the DID–Dyn2 label
at the Seh1–Nup85 arm and Nup120 arm.
a
Nup84 complex
Unmodified
DID1-Seh1 Nup84 complex
No DID–Dyn2 label
1
1
3
3
2
b
2
DID1-Seh1 Nup84 complex with Dyn2 and Flag-DID2
DID-Dyn2 label
1
complex of interest on the first affinity matrix until the final elution
of the sample with the structural tag suitable for electron micro­scopy
analysis, takes about 4 h in minimum. When analyzed by SDSPAGE and Coomassie blue staining, the Nup84 complex carrying
the DID–Dyn2 label showed the expected subunit pattern, including
DID1-Seh1 (or DID1-Nup120), Flag-DID2 and Dyn2 (Fig. 2b and
Supplementary Fig. 1). We then subjected this highly purified Nup84
complex, labeled with DID–Dyn2, to electron microscopy analysis.
Notably, we saw readily observable Y-shaped particles (~20 nm in
diameter) with a ~25-nm rod-like structure protruding from one of
the three arms on negatively stained overview micrographs (Fig. 2c
and Supplementary Fig. 1).
Image processing and topological assignment
By inspecting single particles on the electron microscopy grid, we
could discriminate between the three different arms of the Nup84
complex, and a primary allocation of the DID–Dyn2 label to one of
these three arms was possible (Fig. 2c, gallery). We achieved ­further
clarity regarding the location of the electron microscopy label on
Seh1 or Nup120 when we collected single particles and analyzed
them by image processing and classification. These class averages
revealed a detailed view of the pentameric Y-shaped complex carrying the electron microscopy label on either DID1-Seh1 (Fig. 3b) or
DID1-Nup120 (Fig. 3c), which was highly similar to the unmodified Nup84 complex or the DID1-Seh1–Nup84 complex carrying no
DID–Dyn2 label (Fig. 3a). The feature of the fused label itself (that is,
the ‘pearls-on-a-string’ structure) is less distinct in the classes aligned
to the complex due to a certain flexibility of the label. According
to these classes, the first arm (Fig. 3d, 1) corresponds to the Seh1Nup85 heterodimer with the Seh1 N-terminal end at the arm tip. The
­second arm (Fig. 3d, 2), although not marked with the label, should
correspond to the Sec13-Nup145C heterodimer, since the third arm
(Fig. 3d, 3) carries the electron microscopy label at DID1-Nup120.
Apparently, the N-terminal end of Nup120—the DID1 attachment
site—is not located at the tip end, as the DID–Dyn2 protrudes from
an internally located ‘heel’-like structure that is characteristic for the
third arm (Fig. 3c). Although both electron microscopy labels, on
either Seh1 or Nup120, showed a low level of angular flexibility as
seen by the positioning of the DID–Dyn2 label in different classes,
they nevertheless indicate the exact position of these subunits within
the complex, making even a precise topological mapping of their
respective N termini possible.
DISCUSSION
Here, we present a quick, easy-to-handle and efficient method for
precise subunit labeling within protein complexes by electron microscopy. We have chosen a relatively short ~80-residue label (DID1;
composed of six QT consensus peptides in a row) that can be easily
attached to either terminus of the target protein by DNA recombinant
techniques. Like other QT peptide motifs, DID1 is most likely to
2
3
DID1-Seh1
c
DID1-Nup120 Nup84 complex with Dyn2 and Flag-DID2
DID-Dyn2 label
1
2
3
DID1-Nup120
d
Seh1-Nup85 arm
1
2
3
Nup120 arm
Sec13-Nup145C arm
remain unfolded in its native state. Hence, the QT peptides in an
extended conformation can fit into the two binding grooves of the
dynein light chain homodimer9. Moreover, a short and unfolded tag
fused to a target protein may interfere less by affecting the folding
and assembly of the target subunit into the multimeric complex than
by adding an additional bulky mass (for example, GFP). Such an
unfolded tag may also better protrude from the labeled subunit and
hence can build up in vitro the structural electron microscopy label
by recruiting Dyn2 homodimers and the second Flag-DID2. Crucial
for this method is also the second affinity-purification step using
anti-Flag Sepharose chromatography to exploit the Flag epitope on
DID2. Thus, our method allows for the selective enrichment of the
DID–Dyn2–labeled complex and guarantees that the labeled sample
is sufficiently pure for electron microscopy.
As a proof of principle, we have attached the DID1 to two different
proteins within a given multisubunit complex. The topological map
we obtained revealed the strength of this new electron microscopy
labeling method, which is supported by previous docking studies of
the same complex12. Because the label itself is stable over a large range
of salt conditions, no adaption of the preferred buffer of the complex
of interest should be necessary. One potential drawback, especially for
more globular complexes, may be the length of the label, which could
induce a preferred orientation of the protein on the copper grid. If
this problem developed, the view from several different angles would
be lost. This problem could be overcome by embedding the sample in
vitrified ice and performing cryo–electron microscopy. Also, unlike
antibodies against specific surface epitopes, the labeling of a terminus,
which is located in the center of a complex or is highly flexible, might
hinder a proper allocation. To date, we have shown the suitability of
the label for a complex that was recombinantly expressed in E. coli.
For other expression systems (for example, yeast), a strain without
intrinsic Dyn2 could be chosen to avoid in vivo dimerization (data
not shown).
nature structural & molecular biology advance online publication
TEC H NICAL RE P ORTS
Altogether, it seems that our electron microscopy marker is suitable
for both single proteins and protein (or RNP) complexes and allows
topological information and assignation of the position of either
the N or C terminus of a protein to be gained by electron microscopy. As a future goal, we want to expand this method and test more
applications; for example, to simultaneously label two subunits in a
multimeric complex by fusing two Dyn2-binding motifs of different
lengths to two different subunits. Mapping the relative position of
two subunits with respect to each other may eventually allow the
dynamic behavior of proteins within macromolecular assemblies to
be determined.
Methods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/nsmb/.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
Acknowledgments
We thank P. Bork and C. Müller for providing the facilities for transmission
electron microscopy at the EMBL Heidelberg and M. Lutzmann (Institute of
Human Genetics) for creating the pET24d-NUP85–SEH1 and pPROEXHtbGST-TEV-NUP145C–SEC13-T7-NUP120 expression plasmids. K.T. is a recipient
of the Kekulé grant from Fonds der Chemischen Industrie. E.H. is a recipient of
grants from the Deutsche Forschungsgemeinschaft (SFB 638/B2) and Fonds der
Chemischen Industrie.
AUTHOR CONTRIBUTIONS
D.F., P.S. and E.H. initiated the project; D.F., K.T. and P.S. designed and performed
the experiments; B.B. contributed to electron microscopy image processing and
discussion; E.H. supervised the project.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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advance online publication nature structural & molecular biology
ONLINE METHODS
Construction of DID1 and Flag-DID2 and cloning into expression plasmids.
We engineered DID1 by joining three tandem ‘QT’ 11-mer peptides from Pac11
(dynein intermediate chain; residues 46–57 joined with residues 76–87) yielding six Dyn2-binding motifs in a row (Fig. 1, DID1). We cloned DID1 into the
BamH1/EcoR1 sites of the pProEX-HTb-GST-TEV vector and yeast DYN2
as a NdeI/BamHI fragment into a modified pet24d+ (Novagen) vector with a
N-terminal histidine tag. We used the pProEX-HTb-GST-TEV-Flag-DID construct8 and performed microbiological techniques (growth and transformation of
Escherichia coli) and standard DNA manipulations (cloning, PCR amplification
and ligation) as described previously10.
Further, we cloned a bi-cistronic expression construct consisting of NUP85
­followed by SEH1 as a NheI/BamHI fragment into pET24d (Novagen) and
achieved the N-terminal attachment of the DID1 sequence by blunt-end cloning into a generated ScaI restriction site after the start codon of SEH1 in the
described vector. For the labeling of NUP120 with DID1, we first inserted NUP120
as NsiI/NsiI fragment into the vector pProEX-HT-GST-TEV-NUP145C–SEC13
(described in ref. 10) but carrying its own T7 promoter and terminator. Then we
performed the N-terminal attachment of DID1 to NUP120 by cloning DID1 DNA
as NdeI/NdeI fragment in frame after the start codon of NUP120.
Expression and purification of proteins. We expressed recombinant DYN2
and Flag-DID2 in BL21 codon plus RIL cells (Stratagene) at 23 °C for 3 h in
minimal medium and induced them with 0.5 mM IPTG. We expressed the
DID1-labeled or unlabeled pentameric complex NUP85–SEH1–NUP120-GSTTEV-NUP145C–SEC13 in E. coli overnight at 16 °C in minimal medium and
induced it with 0.5 mM IPTG. After harvesting the cells by centrifugation at
5,000g, we washed the cells in cold water, froze them in liquid nitrogen and
stored the pellets at −20 °C. We performed protein purification of Flag-DID2
as previously described8 and removed the histidine-tagged TEV-protease after
the TEV cleavage by adding TALON Metal Affinity Resin (Clontech) for 30 min
at 4 °C. We lysed the cells expressing the Nup84 complex in buffer containing
300 mM NaCl, 150 mM potassium acetate, 20 mM Tris, pH 7.5, 2 mM magnesium
acetate, 10% (v/v) glycerol and 0.1% (v/v) Nonidet P-40 with a microfluidizer and
incubated the supernatant after centrifugation with glutathione-Sepharose 4B
beads (Amersham/Pharmacia) for 2 h at 4 °C. Subsequently, we washed the beads
with 20 volumes of lysis buffer and 20 volumes of NB buffer containing 150 mM
doi:10.1038/nsmb.1811
NaCl, 50 mM potassium acetate, 20 mM Tris, pH 7.5, 2 mM magnesium acetate
and 0.01% Nonidet P-40. To develop the electron microscopy label, we incubated
the glutathione-Sepharose beads carrying the immobilized Nup84 complex with
either DID1-Seh1 or DID1-Nup120 with an excess of purified Dyn2 and FlagDID2 for 1 h or 16 h at 4 °C. After extensive washing with NB buffer, we performed TEV cleavage and bound the TEV eluate to anti-Flag Sepharose (Sigma)
before elution with Flag peptides according to the manufacturer’s instructions. In
the case of the Nup84 complex without Dyn2–Flag-DID2 bound, we purified the
complex to homogeneity by size-exclusion chromatography in NB buffer using
an Äkta-Basic-System (Amersham/Pharmacia) over a Superose 6 30/10 column
(Amersham/Pharmacia). During gel filtration, we collected 0.6-ml fractions and
analyzed them by SDS-PAGE. Finally, we used single fractions containing the
highly purified complex for electron microscopy.
Electron microscopy and image processing. For negative staining, we placed
6 μl of sample on a freshly glow-discharged, carbon-coated grid and then washed
it three times with water, stained it with uranyl acetate (2% w/v) and dried it. We
recorded the shown micrographs with a Morgagni (FEI) equipped with a 1K side
mounted camera (SIS) operating at 100 kV and micrographs for image processing
with a Phillips CM-200 FEG microscope under low-dose conditions on a 2K ×
2K Tietz-CCD camera (TVIPS F224) at 200 kV with a nominal magnification of
27,500× (calibrated pixel size 5.19 Å per pixel). For image processing, we manually selected a total of 2,088 (for the DID1–Dyn2–DID2 complex), 1,426 (for the
Nup84 complex), 1,226 (for Seh1–Nup84 without Dyn2), 2,625 (for the labeled
Seh1–Nup84 complex) and 1,748 (for the labeled Nup120–Nup84 complex) particles using boxer (EMAN13) with box sizes of 100 × 100 pixels and 128 × 128 pixels,
respectivly. We carried out all subsequent image processing in Imagic V (Image
Science Software GmbH14) and followed previously described procedures15 for
alignment and iterative refinement of class averages.
13.Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for
high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97
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processing system. J. Struct. Biol. 116, 17–24 (1996).
15.Lutzmann, M. et al. Reconstitution of Nup157 and Nup145N into the Nup84
complex. J. Biol. Chem. 280, 18442–18451 (2005).
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