RESEARCH ARTICLE
STRUCTURAL BIOLOGY
Atomic-resolution structure of cytoskeletal
bactofilin by solid-state NMR*
2015 © The Authors, some rights reserved;
exclusive licensee American Association for
the Advancement of Science. Distributed
under a Creative Commons Attribution
NonCommercial License 4.0 (CC BY-NC).
10.1126/sciadv.1501087
Chaowei Shi,1† Pascal Fricke,1† Lin Lin,2,3‡ Veniamin Chevelkov,1 Melanie Wegstroth,4 Karin Giller,4 Stefan Becker,4
Martin Thanbichler,2,3,5 Adam Lange1,6§
The cytoskeleton was once thought to be a unique feature of eukaryotic cells, but homologs to all major components of the eukaryotic cytoskeleton have also been found in prokaryotes (1–3). Additionally,
bacteria-specific cytoskeletal elements have been identified. Among
them are the bactofilins, a recently discovered family of cytoskeletal
proteins that occur in most bacterial lineages (4–6). Bactofilins have
a central conserved domain and variable proline-rich flanking terminal regions. They polymerize spontaneously and independently of
nucleotides or other cofactors and are involved in a variety of crucial
cellular processes. In the prosthecate a-proteobacterium Caulobacter
crescentus, the two bactofilin paralogs BacA and BacB assemble into
membrane-associated polymeric sheets that are specifically localized to
the cell pole carrying the stalk—a thin protrusion of the cell body involved in cell attachment and nutrient acquisition (4). These assemblies serve as spatial landmarks mediating the polar localization of the
cell wall biosynthetic enzyme PbpC, which is involved in stalk biogenesis and organization. The human pathogen Helicobacter pylori,
by contrast, requires the bactofilin homolog CcmA for maintaining its
characteristic helical cell shape, a feature required for cells to efficiently
colonize the gastric mucus (7).
Recently, we demonstrated that high-quality solid-state nuclear magnetic resonance (ssNMR) spectra of BacA could be obtained, which
allowed us to achieve the resonance assignment for the central core domain (residues 37 to 139) (8). As determined from conformationdependent chemical shifts (9), BacA adopts a structure with a rigid core
comprising 18 b strands and flexible N- and C-terminal regions. On
the basis of the assigned b strand segments and additional data on the
mass per length of the BacA filaments from scanning transmission
1
Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125
Berlin, Germany. 2Prokaryotic Cell Biology Group, Max Planck Institute for Terrestrial
Microbiology, 35043 Marburg, Germany. 3Faculty of Biology, Philipps-Universität, 35043
Marburg, Germany. 4Department of NMR-Based Structural Biology, Max Planck Institute
for Biophysical Chemistry, 37077 Göttingen, Germany. 5LOEWE Center for Synthetic Microbiology, Philipps-Universität, 35043 Marburg, Germany. 6Institut für Biologie, HumboldtUniversität zu Berlin, 10115 Berlin, Germany.
*Presented in part at the Gordon Research Conference “Computational Aspects—
Biomolecular NMR” (Il Ciocco, 7 to 12 June 2015).
†These authors contributed equally to this work.
‡Present address: Focal Area Infection Biology, Biozentrum, University of Basel,
Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
§Corresponding author. E-mail: [email protected]
Shi et al. Sci. Adv. 2015;1:e1501087
4 December 2015
electron microscopy, a b-helical fold was proposed for the BacA subunit
(8). Additionally, homology modeling of BacA (8) and its Myxococcus
xanthus homolog BacM (10) suggested a left-handed b-helical arrangement. Because no cytoskeletal filament protein has yet been reported
to adopt a b-helical fold, the determination of an atomic-resolution
structure of a member of the bactofilin family was highly desirable.
X-ray crystallography and solution NMR are difficult to apply to bactofilins because of the rapid polymerization of the subunits into fibrillar
structures during overproduction. In light of the high-quality ssNMR data
reported so far (8) and the recent success in using ssNMR to solve the
atomic structures of supramolecular protein assemblies (11–13), we therefore set out to determine the structure of the BacA subunit by ssNMR.
First, we analyzed spectra of sparsely 13C-labeled BacA samples obtained by growing BacA-overproducing Escherichia coli cells in media
that contained either [1,3-13C]glycerol or [2-13C]glycerol as the sole
carbon source (14, 15). The resulting samples enabled the acquisition
of carbon-carbon correlation spectra of excellent quality and resolution (see figs. S1 and S2). The [2-13C]glycerol–labeled BacA sample
yielded spectra containing many cross peaks in the aromatic and Ca
regions (fig. S1) that could be unambiguously assigned and converted
into long-range (that is, |i − j| > 4) distance restraints. Similarly, spectra
from the [1,3-13C-glycerol; U-15N]–labeled BacA sample provided
long-range distance restraints between side-chain methyl groups (fig.
S2). On the basis of a total of 374 reliable 13C-13C long-range distances
and additional 172 torsion angle restraints, predicted from the assigned
backbone atom chemical shifts (table S1) by the software TALOS-N
(16), a b-helical structure with six windings was calculated. Although
right-handed models were more abundantly generated by the structure
calculations using Xplor-NIH (17), both left-handed (1 to 2 of 20) and
right-handed (18 to 19 of 20) b helices were among the 10% (20 of
200) of the lowest-energy structures.
To unambiguously identify the handedness of the b helix, we collected short [that is, d(H,H) ≤ 5 Å] proton-proton distance restraints
from four-dimensional (4D) HN(H)(H)NH (18, 19) and 2D NHHC
(20) spectra. The excellent resolution of the cross peaks in the protondetected 1H-15N correlation NMR spectrum (Fig. 1A) of deuterated
and 100% back-exchanged BacA37–139 allowed the unambiguous
identification of HN-HN distance restraints from a 4D spectrum. Initially, a total of 96 amide proton resonances were assigned using a
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Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been
reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not
have a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study by
x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and
insolubility. We present the atomic structure of BacA calculated from solid-state NMR–derived distance restraints.
We show that the core domain of BacA forms a right-handed b helix with six windings and a triangular hydrophobic
core. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basis
of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.
RESEARCH ARTICLE
B
A
T110
G51
105
G66
G98
M124
I117
V68
R93
I60
L41 I48 D61
V105
9.5
D45
S40
A89
S111 E120
V90 K136
Y108
A87
R72
E88
F137
V52
L46
T74 R106 A99
A128
Y112
K103
L73
A123
R95
L135
L58
9.0
8.5
1
V90
O
R72
L73
O
H
8.0
7.5
7.0
H chemical shift (ppm)
6.5
9.5
9.0
8.5
1
8.0
7.5
7.0
6.5
E57
C
CA
N
R
H
C
O
T74
O
R
C
CA
CA
N
R
H
L58
O
R
CA
N
C
C
E91
O
H
CA
N
E91
H
L73
R
L58
A39
R
N
C
H
R72
N
H
CA
N
CA
CA
V90
R
Q104
D67
A44
F129 Q59
V97
D114
E91
V113 Y86
E49
E82
R69
F130
T118
O
H
R
C
N
C
A89
V70
E55
I100
E77 T47
S102
V92
V85
V95
G62
O
H
CA
N
G71
G115 G83
G50
S84
G76
Q121
130
R
G79
G54
115
15
N chemical shift (ppm)
110
125
C
2D plane from 4D (L73):
(1H) = 8.55 ppm
3
(15N) = 125.67 ppm
4
T78
G56
120
the 172 torsion angle restraints predicted by TALOS-N and an additional 116 b sheet hydrogen bond restraints were used in the NMR structure calculation of the rigid central domain (residues 37 to 139),
resulting in the structure depicted in Fig. 2. The heavy-atom average
root mean square deviation from the mean structure of the 20 lowestenergy conformers out of 200 calculated structures is 0.4 Å for the
backbone and 1.0 Å for all heavy atoms, indicating a high precision
of the calculated structure ensemble.
The overall organization of the BacA core domain is a right-handed
b helix with six windings and a triangular hydrophobic core. The core
is defined by three b strands per winding that form continuous parallel
b sheets (Fig. 2C). Many of the “corners” of the b helix are formed by
glycines, a feature that is commonly seen in b-helical structures. These
glycines are highly conserved within the bactofilin family (8). The b
sheet arrangement is stabilized by hydrogen bonds between adjacent
strands and hydrophobic side chains tightly packed in the interior of
the b helix. All charged residues face toward the outside, and some of
them are arranged on top of each other such that their charges mutually compensate each other (E49-K65-E82, E57-R72, E88-K103-E120,
and D116-R133). The first (#1) and the last (#6) windings of the b helix
are not as well defined by the data as windings #2 to #5. Here, we could
not detect any intermolecular distance restraints between windings #1
and #6 as would be expected from a head-to-tail arrangement of the
subunits. The lack of such restraints could either indicate a different
supramolecular arrangement or reflect an increased local mobility of
windings #1 and #6. All of our observed distance restraints were unambiguously assigned to intrasubunit contacts, leaving no unassigned
restraints that could have arisen from intermolecular subunit-subunit
contacts. This suggests that the lack of restraints for windings #1 and
C
O
Q59
H chemical shift (ppm)
Fig. 1. Structural restraints from 1H-detected ssNMR. (A) 2D H-N correlation spectrum of deuterated and 100% back-exchanged BacA37–139 with some
of the amino acid residues annotated. (B) One plane of the 4D HN(H)(H)NH correlation spectrum. Plane taken at chemical shift positions of the L73 residue
(diagonal peak marked in red). Long-distance restraints (cross peaks) annotated in black. For better visualization, the corresponding residues are labeled in
blue in (A). The appearing cross peaks represent spatially close amide protons and are used for structure calculation. (C) Schematic representation of
magnetization transfer from residue L73 to spatially close residues during the 4D NMR experiment (blue arrows). Dashed red lines represent hydrogen
bonds between consecutive windings of the b helix.
Shi et al. Sci. Adv. 2015;1:e1501087
4 December 2015
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series of 3D proton-detected NMR experiments (21). The 4D HN(H)
(H)NH experiment used 5.2-ms radio frequency–driven recoupling
(RFDR) mixing (22) for the magnetization transfer between adjacent
NH pairs (fig. S3). To reduce the measurement time of the 4D experiment, the data were acquired by sine-weighted Poisson-gap nonuniform sampling (NUS) of 25% of the total number of data points
(23, 24). In the resulting spectrum, the chemical shifts of HNi, Ni, Nj,
and HNj were correlated on the basis of the HNi-HNj dipolar interaction. For example, in the 2D plane defined by the HN and N chemical shifts of residue L73, cross peaks were exclusively observed to
residues spatially close to L73 (Fig. 1, B and C). The distance restraints
collected by this method agreed well with the b helix model generated
by our earlier calculations based on carbon-carbon distances and predicted dihedral angles (see above). Because the peaks are very well
separated in the 4D spectrum, this approach significantly reduces assignment ambiguities. A total of 59 unambiguous HNi-HNj distance
correlations (present in both of the corresponding HN correlation
planes) were extracted (table S2). Structure calculations (see below)
showed that these restraints fitted only to the model of the right-handed
b helix. These results could be further corroborated by HNi-Haj contacts extracted from an NHHC spectrum recorded on the [1,3-13Cglycerol; U-15N]–labeled BacA sample (fig. S4).
In total, 1932 distance restraints were collected, including 178
medium-range (that is, |i − j| = 2, 3, or 4) and 488 long-range contacts
(fig. S5). These peaks were selected in an iterative manner: Before the
first structure calculation, only unambiguous peaks were selected. The
resulting structure was then used to select additional (structurally unambiguous) peaks for refinement. This was repeated in several rounds
until almost all of the peaks in the spectra were assigned. Additionally,
RESEARCH ARTICLE
A
C
Winding 1
Winding 2
L
G
E
S
55
40
V
D
T
D
5
E 125
L*
L
M*
A
122
S
135
R
G
F*
G
G
F
130
Q
Q
K
V
E
120
H
D
L
V 75
G
V V
Y
T
H
85
S
65
105
Y
80
E
G
L
G115
A
G
T
S
I V
T
A
E
Winding 4
R
T
E
V
Winding 5
A
K
G
60
V
V
E
G
110
Y
D
K
E
S
V
T
90
I
A
R
G
V
R
V
95
100
G
V
Fig. 2. Structure of the BacA subunit (residues 37 to 139). (A and B) Top view (A) and side view (B) of BacA. (C) Schematic representation of the six
windings. Hydrophobic residues are colored white, acidic residues are colored red, basic residues are colored blue, and others are colored green. Mutations of residues L122, M124, and F130 (winding #6) to alanine or serine affect the BacA assembly in vivo. These residues are marked with an asterisk.
#6 results from increased dynamic motion in these regions. This possibility is further supported by the observation that cross-peak intensities in the dipolar-based correlation spectra used for the assignment
were generally attenuated for residues located within these windings
(fig. S6). Notably, mutations in winding #6 (L122S, M124A, M124S,
and F130A) (8) were previously shown to affect BacA assembly in vivo,
whereas an A123S exchange did not. On the basis of the arrangement of
winding #6 determined here, the side chains of L122, M124, and F130
point to the hydrophobic core, whereas that of A123 points outside (see
Fig. 2C). Mutation of the three core residues may thus destabilize
winding #6 or render the surface provided by winding #6 less hydrophobic, thereby potentially preventing intersubunit interaction.
Here, we have determined a de novo atomic-resolution structure of
the bactofilin BacA, on the basis of a large number of unambiguous
ssNMR-derived distance restraints. Thus far, the right-handed b-helical
fold has not been described for any other cytoskeletal protein. However,
the BacA structure is similar to that of the prion protein HET-s in its
amyloid state (11). It will be interesting in the future to investigate the
arrangement of BacA in the context of the membrane-associated polymeric sheets observed in vivo by complementary techniques such as
cryo-electron microscopy and tomography (25–27) and to determine
a hybrid structure of the whole assembly (28, 29).
MATERIALS AND METHODS
Sample preparation
To prepare 13C-labeled samples, we produced BacA-His6 in M9 medium with 0.3% (v/v) [2-13C]glycerol or [1,3-13C]glycerol as the sole
Shi et al. Sci. Adv. 2015;1:e1501087
4 December 2015
carbon source. The [1,3-13C]glycerol–containing medium additionally
contained 15NH4Cl as the sole nitrogen source. The overproduction
protocol was based on a previously described procedure (8). Briefly, cells
of E. coli Rosetta(DE3)pLysS (Invitrogen) were transformed with pMT879
and grown in 1 liter of SB medium supplemented with appropriate
antibiotics at 37°C to an OD600 (optical density at 600 nm) of 3 to 4.
Cells were harvested by centrifugation and washed with 100 ml of M9
salt solution. The pellets were then resuspended in 1 liter of M9 medium containing the indicated carbon and nitrogen sources. The cultures were grown for 1.5 hours at 37°C. Subsequently, the production
of BacA-His6 was induced by the addition of 0.5 mM isopropyl-b-Dthiogalactopyranoside (IPTG). After 24 hours of cultivation, the cells
were harvested, and BacA-His6 was purified as described previously. The
samples, together with a few crystals of DSS (4,4-dimethyl-4-silapentane1-sulfonic acid), were packed into 4-mm magic angle spinning (MAS)
rotors, using protein quantities of ~30 mg each.
To produce a shortened version of BacA that corresponded to the
rigid DUF583 domain of BacA, we cloned the coding sequence for
amino acids 37 to 139 into a modified pET28a vector (Clontech), encoding a cleavable N-terminal heptahistidine tag. After transformation
into E. coli BL21(DE3), cells were grown at 37°C in an M9 minimal medium with 15NH4Cl and 13C6-D-glucose as sole nitrogen and carbon
sources, respectively, prepared in 99.9% D2O. Overproduction of the
[2H,13C,15N]-labeled fusion protein was induced by the addition of
0.5 mM IPTG. The culture was harvested 15 hours after induction,
resuspended in lysis buffer [50 mM tris-HCl (pH 8.0), 500 mM NaCl,
5 mM imidazole, and 0.5 mM phenylmethylsulfonyl fluoride] supplemented with Benzonase (25 U/ml; Merck Millipore), and lysed by
sonication. From the supernatant, the fusion protein was purified by
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2
3
4
6
I
70
R
Winding 6
Q
V
T
D
L
G
45
I
G50
R
Q
A
L
G
1
E
L
G
T
B
S
Winding 3
RESEARCH ARTICLE
immobilized metal affinity chromatography on Ni-NTA (nickel–
nitrilotriacetic acid) Sepharose. The protein was eluted with 20 column
volumes of buffer with 300 mM imidazole. The eluate was dialyzed
against tobacco etch virus (TEV) cleavage buffer [50 mM tris-HCl (pH
8.0), 0.5 mM EDTA, and 1 mM dithiothreitol], and the fusion tag was
cleaved with 1 mg of TEV per 100 mg of fusion protein overnight at
room temperature before removal with Ni-NTA Sepharose. The concentration of the protein was adjusted to 1.5 mg/ml, and the sample was
kept at room temperature for 8 days. The resulting aggregate, formed
from a total of 42 mg of protein, was collected by centrifugation. The
sample was transferred into a 1.9-mm NMR rotor equipped with a bottom spacer. Before closing the rotor, a few crystals of DSS and about
1 ml of D2O were added.
Shi et al. Sci. Adv. 2015;1:e1501087
4 December 2015
Structure calculation and validation
Backbone f and y torsion angle restraints were predicted from the
patterns of the backbone atom chemical shifts using the TALOS-N
software package. Assigned ssNMR cross peaks from the PDSD spectra
were manually converted into internuclear carbon-carbon distance restraints with a single distance range of 1 to 8 Å. The information extracted from NHHC and 4D HN(H)(H)NH spectra was converted
into interproton distance restraints with a distance range of 1 to 6 Å
(fig. S7). Hydrogen bond donor and acceptor atoms used for the final
structure refinement were identified on the basis of the secondary structure elements predicted by TALOS-N and by manual inspection of
earlier rounds of structures.
All structure calculations were performed using Xplor-NIH version
2.37. With an extended template structure, calculations were initiated
by randomizing the backbone torsion angles and then running 50 cycles of Powell energy minimization to remove close nonbonded contacts. Simulated annealing was performed in two stages, using 20,000
steps at 1500 K and 30,000 steps with gradual cooling to 100 K using
a constant time step of 5 fs. Finally, the annealed structures were
energy-minimized using a 200-step Powell energy minimization. The
20 lowest-energy structures out of 200 calculated structures were
selected.
Visualization was performed using PyMOL (37). Atomic coordinates and structure constraints were deposited in the Protein Data Bank
(PDB ID: 2N3D). The quality of the final ensemble of 20 NMR structures was evaluated by submission to the Protein Structure Validation
Software suite version 1.5 (38). A summary of the restraints used in
the final structure calculations and the global structure quality factors is
provided in table S4.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/1/11/e1501087/DC1
Fig. S1. 2D 13C-13C PDSD spectrum of [2-13C]glycerol–labeled BacA.
Fig. S2. 2D 13C-13C PDSD spectrum of [1,3-13C-glycerol; U-15N]–labeled BacA.
Fig. S3. Schematic representation of the 4D HN(H)(H)NH sequence.
Fig. S4. 2D NHHC spectrum of [1,3-13C-glycerol; U-15N]–labeled BacA.
Fig. S5. BacA sequence and distance restraints used for structure calculation.
Fig. S6. Intensities of the cross peaks in the 3D (H)CANH spectrum.
Fig. S7. Schematic representation of the approximate amide proton–amide proton distances
within the parallel b sheets.
Table S1. Assigned chemical shifts of BacA (in ppm, relative to internal DSS).
Table S2. Summary of the distance restraints collected from the 4D HN(H)(H)NH spectrum.
Table S3. Experimental details.
Table S4. BacA ssNMR structure calculation statistics.
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NMR spectroscopy and detection of distance restraints
Carbon-detected ssNMR experiments were conducted on 16.4- and
18.8-T (1H resonance frequencies of 700 and 800 MHz, respectively)
wide-bore NMR spectrometers (Bruker Biospin) equipped with 4-mm
triple-resonance (1H, 13C, and 15N) MAS probes. Spectra were recorded
at an MAS rate of 11 kHz and were calibrated with DSS as an internal
proton chemical shift reference. The sample temperature was kept
constant at about 4°C, as measured by the temperature-dependent position of the water resonance peak with respect to internal DSS (30).
High-power 1H-13C decoupling with a radio-frequency amplitude of
about 80 kHz was applied during evolution and detection periods (31).
To detect medium- and long-range contacts, we used the proton-driven
spin diffusion (PDSD) scheme with a longitudinal mixing time of
800 ms for BacA produced with [2-13C]glycerol and mixing times
of 300 and 700 ms for BacA produced with [1,3-13C]glycerol. An
NHHC (20) spectrum with a (1H,1H) mixing time of 200 ms was recorded at 18.8 T using BacA produced with [1,3-13C]glycerol. Contact
times of tNH = 300 ms and tHC = 90 ms were used to ensure polarization transfer within NH or CHx (x = 1, 2, or 3) groups only.
The 1H-detected NMR spectra were acquired on a Bruker 21.2-T
(900-MHz 1H Larmor frequency) standard-bore NMR magnet equipped
with a 1.9-mm, four-channel (1H,2H,13C,15N) probe (32). The MAS frequency was set to 40 kHz, and the sample temperature was set to 28°C.
The field was locked to the deuterium resonance resulting from the
added D2O.
Backbone chemical shift assignment was achieved using five 3D
NMR spectra: (H)CANH, (H)CONH, (H)CACO(N)H, (H)COCA(N)H,
and (H)CA(CO)NH. The used assignment strategy and experimental
details closely follow the protocols described earlier (21, 33, 34). The
long-distance restraints were collected using a 4D NMR [HN(H)(H)
NH] spectrum with RFDR mixing (22) for the magnetization transfer
between spatially adjacent NH pairs. RFDR inversion pulses with a radiofrequency amplitude of 100 kHz were applied. The RFDR mixing time
was 5.2 ms. 1H, 13C, and 15N chemical shifts are listed in table S1 and
were deposited into the Biological Magnetic Resonance Data Bank
(BMRB entry: 25642). An overview of the experimental details is
provided in table S3.
To reduce the measurement time of the 4D NMR spectrum from
~36 to ~9 days, the data were acquired by sine-weighted Poisson-gap
NUS of 25% of the data points (23, 24). The reconstruction of the missing
free induction decays (FIDs) was carried out with the iterative softthreshold method implemented in the hmsIST software. Spectral processing was carried out with NMRPipe 8.2 (35). A squared sine bell
window function with a shift of p/3 was applied to the 512 acquired
complex points of the FIDs. After zero-filling to 1024 complex points,
the FIDs were Fourier-transformed and the region between 10 and
5 parts per million (ppm) was extracted. The three indirect dimensions
were then reconstructed by hmsIST to 64 complex points for each
dimension, with 3000 iterations and a level multiplier of 0.995. A squared
sine bell window function with a shift of 2p/3 was applied to each indirect dimension. Subsequent Fourier transform was carried out after
zero-filling to 128 complex points for each indirect dimension. A
phase correction was only applied for the direct dimension. The resulting NMR spectrum was viewed and assigned in CcpNmr Analysis
version 2.4 (36). Chemical shift referencing was carried out by overlaying the referenced 2D H-N correlation spectrum.
RESEARCH ARTICLE
REFERENCES AND NOTES
Shi et al. Sci. Adv. 2015;1:e1501087
4 December 2015
Acknowledgments: We thank L. Ball for discussions regarding the structure calculation and
A. Nieuwkoop and M. Hiller for technical help. Funding: This work was supported by the LeibnizInstitut für Molekulare Pharmakologie, the Max Planck Society, the European Research Council
(ERC Starting Grant to A.L.), the German Research Foundation (Deutsche Forschungsgemeinschaft;
Emmy Noether Fellowship to A.L. and support from Research Training Group GRK 1216 to L.L.), the
Fonds der Chemischen Industrie (Kekulé Scholarship to P.F.), and the Human Frontier Science
Program (Young Investigator Grant RGY0076/2013 to M.T.). Author contributions: C.S. and P.F.
performed ssNMR experiments. C.S., P.F., and A.L. analyzed NMR data. C.S. performed structure
calculations. L.L., M.W., K.G., S.B., and M.T. expressed, purified, and polymerized BacA samples. C.S.,
P.F., and A.L. wrote the report. All authors discussed the results and commented on the manuscript.
Competing interests: The authors declare that they have no competing interests. Data and materials
availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or
the Supplementary Materials. Additional data related to this paper may be requested from the authors.
The BacA chemical shifts were deposited in the BMRB under accession code 25642, and its structure
was deposited in the PDB under accession code 2N3D.
Submitted 12 August 2015
Accepted 15 October 2015
Published 4 December 2015
10.1126/sciadv.1501087
Citation: C. Shi, P. Fricke, L. Lin, V. Chevelkov, M. Wegstroth, K. Giller, S. Becker, M. Thanbichler,
A. Lange, Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR. Sci. Adv. 1,
e1501087 (2015).
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Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR
Chaowei Shi, Pascal Fricke, Lin Lin, Veniamin Chevelkov, Melanie Wegstroth, Karin Giller, Stefan Becker, Martin Thanbichler
and Adam Lange
Sci Adv 1 (11), e1501087.
DOI: 10.1126/sciadv.1501087
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