atomic-resolution structure of cytoskeletal bactofilin by solid ......the baca structure was...

R E S EARCH ART I C L E

STRUCTURAL B IOLOGY

1Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125Berlin, Germany. 2Prokaryotic Cell Biology Group, Max Planck Institute for TerrestrialMicrobiology, 35043 Marburg, Germany. 3Faculty of Biology, Philipps-Universität, 35043Marburg, Germany. 4Department of NMR-Based Structural Biology, Max Planck Institutefor Biophysical Chemistry, 37077 Göttingen, Germany. 5LOEWE Center for Synthetic Mi-crobiology, Philipps-Universität, 35043 Marburg, Germany. 6Institut für Biologie, Humboldt-Universität 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

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10.1126/sciadv.1501087

Atomic-resolution structure of cytoskeletalbactofilin by solid-state NMR*

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§

D

Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has beenreported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morpho-genesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do nothave a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study byx-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity andinsolubility. 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 hydrophobiccore. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basisof unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.

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The cytoskeleton was once thought to be a unique feature of eukaryo-tic cells, but homologs to all major components of the eukaryotic cyto-skeleton have also been found in prokaryotes (1–3). Additionally,bacteria-specific cytoskeletal elements have been identified. Amongthem are the bactofilins, a recently discovered family of cytoskeletalproteins that occur in most bacterial lineages (4–6). Bactofilins havea central conserved domain and variable proline-rich flanking ter-minal regions. They polymerize spontaneously and independently ofnucleotides or other cofactors and are involved in a variety of crucialcellular processes. In the prosthecate a-proteobacterium Caulobactercrescentus, the two bactofilin paralogs BacA and BacB assemble intomembrane-associated polymeric sheets that are specifically localized tothe cell pole carrying the stalk—a thin protrusion of the cell body in-volved in cell attachment and nutrient acquisition (4). These assem-blies serve as spatial landmarks mediating the polar localization of thecell wall biosynthetic enzyme PbpC, which is involved in stalk bio-genesis and organization. The human pathogen Helicobacter pylori,by contrast, requires the bactofilin homolog CcmA for maintaining itscharacteristic helical cell shape, a feature required for cells to efficientlycolonize the gastric mucus (7).

Recently, we demonstrated that high-quality solid-state nuclear mag-netic resonance (ssNMR) spectra of BacA could be obtained, whichallowed us to achieve the resonance assignment for the central core do-main (residues 37 to 139) (8). As determined from conformation-dependent chemical shifts (9), BacA adopts a structure with a rigid corecomprising 18 b strands and flexible N- and C-terminal regions. Onthe basis of the assigned b strand segments and additional data on themass per length of the BacA filaments from scanning transmission

electron microscopy, a b-helical fold was proposed for the BacA subunit(8). Additionally, homology modeling of BacA (8) and its Myxococcusxanthus homolog BacM (10) suggested a left-handed b-helical arrange-ment. Because no cytoskeletal filament protein has yet been reportedto adopt a b-helical fold, the determination of an atomic-resolutionstructure of a member of the bactofilin family was highly desirable.X-ray crystallography and solution NMR are difficult to apply to bacto-filins because of the rapid polymerization of the subunits into fibrillarstructures during overproduction. In light of the high-quality ssNMR datareported so far (8) and the recent success in using ssNMR to solve theatomic structures of supramolecular protein assemblies (11–13), we there-fore set out to determine the structure of the BacA subunit by ssNMR.

First, we analyzed spectra of sparsely 13C-labeled BacA samples ob-tained by growing BacA-overproducing Escherichia coli cells in mediathat contained either [1,3-13C]glycerol or [2-13C]glycerol as the solecarbon source (14, 15). The resulting samples enabled the acquisitionof carbon-carbon correlation spectra of excellent quality and resolu-tion (see figs. S1 and S2). The [2-13C]glycerol–labeled BacA sampleyielded spectra containing many cross peaks in the aromatic and Caregions (fig. S1) that could be unambiguously assigned and convertedinto long-range (that is, |i − j| > 4) distance restraints. Similarly, spectrafrom the [1,3-13C-glycerol; U-15N]–labeled BacA sample providedlong-range distance restraints between side-chain methyl groups (fig.S2). On the basis of a total of 374 reliable 13C-13C long-range distancesand additional 172 torsion angle restraints, predicted from the assignedbackbone atom chemical shifts (table S1) by the software TALOS-N(16), a b-helical structure with six windings was calculated. Althoughright-handed models were more abundantly generated by the structurecalculations using Xplor-NIH (17), both left-handed (1 to 2 of 20) andright-handed (18 to 19 of 20) b helices were among the 10% (20 of200) of the lowest-energy structures.

To unambiguously identify the handedness of the b helix, we col-lected short [that is, d(H,H) ≤ 5 Å] proton-proton distance restraintsfrom four-dimensional (4D) HN(H)(H)NH (18, 19) and 2D NHHC(20) spectra. The excellent resolution of the cross peaks in the proton-detected 1H-15N correlation NMR spectrum (Fig. 1A) of deuteratedand 100% back-exchanged BacA37–139 allowed the unambiguousidentification of HN-HN distance restraints from a 4D spectrum. Ini-tially, a total of 96 amide proton resonances were assigned using a

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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 adjacentNH pairs (fig. S3). To reduce the measurement time of the 4D exper-iment, the data were acquired by sine-weighted Poisson-gap non-uniform 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 inter-action. For example, in the 2D plane defined by the HN and N chem-ical shifts of residue L73, cross peaks were exclusively observed toresidues spatially close to L73 (Fig. 1, B and C). The distance restraintscollected by this method agreed well with the b helix model generatedby our earlier calculations based on carbon-carbon distances and pre-dicted dihedral angles (see above). Because the peaks are very wellseparated in the 4D spectrum, this approach significantly reduces as-signment ambiguities. A total of 59 unambiguous HNi-HNj distancecorrelations (present in both of the corresponding HN correlationplanes) were extracted (table S2). Structure calculations (see below)showed that these restraints fitted only to the model of the right-handedb helix. These results could be further corroborated by HNi-Haj con-tacts extracted from an NHHC spectrum recorded on the [1,3-13C-glycerol; U-15N]–labeled BacA sample (fig. S4).

In total, 1932 distance restraints were collected, including 178medium-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 thefirst structure calculation, only unambiguous peaks were selected. Theresulting structure was then used to select additional (structurally un-ambiguous) peaks for refinement. This was repeated in several roundsuntil almost all of the peaks in the spectra were assigned. Additionally,


the 172 torsion angle restraints predicted by TALOS-N and an addition-al 116 b sheet hydrogen bond restraints were used in the NMR struc-ture calculation of the rigid central domain (residues 37 to 139),resulting in the structure depicted in Fig. 2. The heavy-atom averageroot mean square deviation from the mean structure of the 20 lowest-energy conformers out of 200 calculated structures is 0.4 Å for thebackbone and 1.0 Å for all heavy atoms, indicating a high precisionof the calculated structure ensemble.

The overall organization of the BacA core domain is a right-handedb helix with six windings and a triangular hydrophobic core. The coreis defined by three b strands per winding that form continuous parallelb sheets (Fig. 2C). Many of the “corners” of the b helix are formed byglycines, a feature that is commonly seen in b-helical structures. Theseglycines are highly conserved within the bactofilin family (8). The bsheet arrangement is stabilized by hydrogen bonds between adjacentstrands and hydrophobic side chains tightly packed in the interior ofthe b helix. All charged residues face toward the outside, and some ofthem are arranged on top of each other such that their charges mu-tually 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 helixare not as well defined by the data as windings #2 to #5. Here, we couldnot detect any intermolecular distance restraints between windings #1and #6 as would be expected from a head-to-tail arrangement of thesubunits. The lack of such restraints could either indicate a differentsupramolecular arrangement or reflect an increased local mobility ofwindings #1 and #6. All of our observed distance restraints were un-ambiguously assigned to intrasubunit contacts, leaving no unassignedrestraints that could have arisen from intermolecular subunit-subunitcontacts. This suggests that the lack of restraints for windings #1 and

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3(1H) = 8.55 ppm

4(15N) = 125.67 ppm

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Fig. 1. Structural restraints from 1H-detected ssNMR. (A) 2D H-N correlation spectrum of deuterated and 100% back-exchanged BacA37–139 with someof 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 inblue in (A). The appearing cross peaks represent spatially close amide protons and are used for structure calculation. (C) Schematic representation ofmagnetization transfer from residue L73 to spatially close residues during the 4D NMR experiment (blue arrows). Dashed red lines represent hydrogenbonds between consecutive windings of the b helix.

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#6 results from increased dynamic motion in these regions. This pos-sibility is further supported by the observation that cross-peak inten-sities in the dipolar-based correlation spectra used for the assignmentwere 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 ofwinding #6 determined here, the side chains of L122, M124, and F130point to the hydrophobic core, whereas that of A123 points outside (seeFig. 2C). Mutation of the three core residues may thus destabilizewinding #6 or render the surface provided by winding #6 less hydro-phobic, thereby potentially preventing intersubunit interaction.

Here, we have determined a de novo atomic-resolution structure ofthe bactofilin BacA, on the basis of a large number of unambiguousssNMR-derived distance restraints. Thus far, the right-handed b-helicalfold has not been described for any other cytoskeletal protein. However,the BacA structure is similar to that of the prion protein HET-s in itsamyloid state (11). It will be interesting in the future to investigate thearrangement of BacA in the context of the membrane-associated poly-meric sheets observed in vivo by complementary techniques such ascryo-electron microscopy and tomography (25–27) and to determinea hybrid structure of the whole assembly (28, 29).

MATERIALS AND METHODS

Sample preparationTo prepare 13C-labeled samples, we produced BacA-His6 in M9 me-dium with 0.3% (v/v) [2-13C]glycerol or [1,3-13C]glycerol as the sole


carbon source. The [1,3-13C]glycerol–containing medium additionallycontained 15NH4Cl as the sole nitrogen source. The overproductionprotocol was based on a previously described procedure (8). Briefly, cellsof E. coli Rosetta(DE3)pLysS (Invitrogen) were transformed with pMT879and grown in 1 liter of SB medium supplemented with appropriateantibiotics 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 M9salt solution. The pellets were then resuspended in 1 liter of M9 me-dium containing the indicated carbon and nitrogen sources. The cul-tures were grown for 1.5 hours at 37°C. Subsequently, the productionof BacA-His6 was induced by the addition of 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After 24 hours of cultivation, the cellswere harvested, and BacA-His6 was purified as described previously. Thesamples, together with a few crystals of DSS (4,4-dimethyl-4-silapentane-1-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 therigid DUF583 domain of BacA, we cloned the coding sequence foramino acids 37 to 139 into a modified pET28a vector (Clontech), en-coding a cleavable N-terminal heptahistidine tag. After transformationinto E. coli BL21(DE3), cells were grown at 37°C in an M9 minimal me-dium with 15NH4Cl and

13C6-D-glucose as sole nitrogen and carbonsources, respectively, prepared in 99.9% D2O. Overproduction of the[2H,13C,15N]-labeled fusion protein was induced by the addition of0.5 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] supple-mented with Benzonase (25 U/ml; Merck Millipore), and lysed bysonication. From the supernatant, the fusion protein was purified by

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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 sixwindings. Hydrophobic residues are colored white, acidic residues are colored red, basic residues are colored blue, and others are colored green. Muta-

tions 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.

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immobilized metal affinity chromatography on Ni-NTA (nickel–nitrilotriacetic acid) Sepharose. The protein was eluted with 20 columnvolumes of buffer with 300 mM imidazole. The eluate was dialyzedagainst tobacco etch virus (TEV) cleavage buffer [50 mM tris-HCl (pH8.0), 0.5 mM EDTA, and 1 mM dithiothreitol], and the fusion tag wascleaved with 1 mg of TEV per 100 mg of fusion protein overnight atroom temperature before removal with Ni-NTA Sepharose. The con-centration of the protein was adjusted to 1.5 mg/ml, and the sample waskept at room temperature for 8 days. The resulting aggregate, formedfrom a total of 42 mg of protein, was collected by centrifugation. Thesample was transferred into a 1.9-mm NMR rotor equipped with a bot-tom spacer. Before closing the rotor, a few crystals of DSS and about1 ml of D2O were added.

NMR spectroscopy and detection of distance restraintsCarbon-detected ssNMR experiments were conducted on 16.4- and18.8-T (1H resonance frequencies of 700 and 800 MHz, respectively)wide-bore NMR spectrometers (Bruker Biospin) equipped with 4-mmtriple-resonance (1H, 13C, and 15N) MAS probes. Spectra were recordedat an MAS rate of 11 kHz and were calibrated with DSS as an internalproton chemical shift reference. The sample temperature was keptconstant at about 4°C, as measured by the temperature-dependent po-sition of the water resonance peak with respect to internal DSS (30).High-power 1H-13C decoupling with a radio-frequency amplitude ofabout 80 kHz was applied during evolution and detection periods (31).To detect medium- and long-range contacts, we used the proton-drivenspin diffusion (PDSD) scheme with a longitudinal mixing time of800 ms for BacA produced with [2-13C]glycerol and mixing timesof 300 and 700 ms for BacA produced with [1,3-13C]glycerol. AnNHHC (20) spectrum with a (1H,1H) mixing time of 200 ms was re-corded at 18.8 T using BacA produced with [1,3-13C]glycerol. Contacttimes of tNH = 300 ms and tHC = 90 ms were used to ensure polariza-tion 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 NMRmagnet equippedwith a 1.9-mm, four-channel (1H,2H,13C,15N) probe (32). The MAS fre-quency was set to 40 kHz, and the sample temperature was set to 28°C.The field was locked to the deuterium resonance resulting from theadded D2O.

Backbone chemical shift assignment was achieved using five 3DNMR spectra: (H)CANH, (H)CONH, (H)CACO(N)H, (H)COCA(N)H,and (H)CA(CO)NH. The used assignment strategy and experimentaldetails closely follow the protocols described earlier (21, 33, 34). Thelong-distance restraints were collected using a 4D NMR [HN(H)(H)NH] spectrum with RFDR mixing (22) for the magnetization transferbetween spatially adjacent NH pairs. RFDR inversion pulses with a radio-frequency amplitude of 100 kHz were applied. The RFDR mixing timewas 5.2 ms. 1H, 13C, and 15N chemical shifts are listed in table S1 andwere deposited into the Biological Magnetic Resonance Data Bank(BMRB entry: 25642). An overview of the experimental details isprovided 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-gapNUS of 25% of the data points (23, 24). The reconstruction of the missingfree induction decays (FIDs) was carried out with the iterative soft-threshold method implemented in the hmsIST software. Spectral pro-cessing was carried out with NMRPipe 8.2 (35). A squared sine bellwindow 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 and5 parts per million (ppm) was extracted. The three indirect dimensionswere then reconstructed by hmsIST to 64 complex points for eachdimension, with 3000 iterations and a level multiplier of 0.995. A squaredsine bell window function with a shift of 2p/3 was applied to each in-direct dimension. Subsequent Fourier transform was carried out afterzero-filling to 128 complex points for each indirect dimension. Aphase correction was only applied for the direct dimension. The re-sulting NMR spectrum was viewed and assigned in CcpNmr Analysisversion 2.4 (36). Chemical shift referencing was carried out by over-laying the referenced 2D H-N correlation spectrum.

Structure calculation and validationBackbone f and y torsion angle restraints were predicted from thepatterns of the backbone atom chemical shifts using the TALOS-Nsoftware package. Assigned ssNMR cross peaks from the PDSD spectrawere manually converted into internuclear carbon-carbon distance re-straints with a single distance range of 1 to 8 Å. The information ex-tracted from NHHC and 4D HN(H)(H)NH spectra was convertedinto interproton distance restraints with a distance range of 1 to 6 Å(fig. S7). Hydrogen bond donor and acceptor atoms used for the finalstructure refinement were identified on the basis of the secondary struc-ture elements predicted by TALOS-N and by manual inspection ofearlier rounds of structures.

All structure calculations were performed using Xplor-NIH version2.37. With an extended template structure, calculations were initiatedby randomizing the backbone torsion angles and then running 50 cy-cles of Powell energy minimization to remove close nonbonded con-tacts. Simulated annealing was performed in two stages, using 20,000steps at 1500 K and 30,000 steps with gradual cooling to 100 K usinga constant time step of 5 fs. Finally, the annealed structures wereenergy-minimized using a 200-step Powell energy minimization. The20 lowest-energy structures out of 200 calculated structures wereselected.

Visualization was performed using PyMOL (37). Atomic coordi-nates and structure constraints were deposited in the Protein Data Bank(PDB ID: 2N3D). The quality of the final ensemble of 20 NMR struc-tures was evaluated by submission to the Protein Structure ValidationSoftware suite version 1.5 (38). A summary of the restraints used inthe final structure calculations and the global structure quality factors isprovided in table S4.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/11/e1501087/DC1Fig. 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 distanceswithin 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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Acknowledgments: We thank L. Ball for discussions regarding the structure calculation andA. Nieuwkoop and M. Hiller for technical help. Funding: This work was supported by the Leibniz-Institut 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.), theFonds der Chemischen Industrie (Kekulé Scholarship to P.F.), and the Human Frontier ScienceProgram (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 structurecalculations. 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 andmaterialsavailability: All data needed to evaluate the conclusions in the paper are present in the paper and/orthe 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 structurewas deposited in the PDB under accession code 2N3D.

Submitted 12 August 2015Accepted 15 October 2015Published 4 December 201510.1126/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

and Adam LangeChaowei Shi, Pascal Fricke, Lin Lin, Veniamin Chevelkov, Melanie Wegstroth, Karin Giller, Stefan Becker, Martin Thanbichler

DOI: 10.1126/sciadv.1501087 (11), e1501087.1Sci Adv

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MATERIALSSUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2015/12/01/1.11.e1501087.DC1

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atomic-resolution structure of cytoskeletal bactofilin by solid ......the baca structure was...

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