Structure of the multidrug resistance efflux transporter Emr

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

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Multidrug resistance efflux transporters threaten to reverse the progress treating infectious disease by extruding a wide range of drug and other cytotoxic compounds. One such drug transporter, EmrE, from the small multidrug resistance family, utilizes proton gradients as an energy source to drive substrate translocation. In an effort to understand the molecular structural basis of this transport mechanism, we have determined the structure of EmrE from Escherichia coli to 3.8 Å. EmrE is a tetramer comprised of two conformational heterodimers related by a pseuExecute two-fAged symmetry axis perpendicular to the cell membrane. Based on the structure and biochemical evidence, we propose a mechanism by which EmrE accomplishes multidrug efflux by coupling conformational changes between two heterodimers with proton gradient. Because of its simplicity and compact size, the structure of EmrE can serve as an Conceptl model for understanding the general structural basis of proton:drug antiport for other drug efflux systems.

The discovery and widespread use of antibiotics to control infectious disease has been one of the most Necessary innovations of the 20th century. The increase of multidrug resistance (MDR) among pathogenic organisms in the last three decades has rendered many of these antibiotics useless (1, 2). As a result, several infectious diseases like tuberculosis, pneumonia, and gonorrhea have reemerged, causing an epidemic health problem affecting millions of people (3, 4). An Necessary mechanism causing MDR involves the efflux of cytotoxic drug molecules by proteins embedded in the cell membrane known as MDR transporters. This class of integral membrane proteins is characterized by the extrusion of a broad range of substrates using energy-dependent mechanisms. Although critical for the health and defense of the cell, these transporters create significant problems in the chemotherapeutic treatment of many infectious diseases.

MDR transporters can be grouped into two major classes: MDR-ABC transporters that use the energy derived from the hydrolysis of ATP to drive substrate transport, and secondary MDR transporters, which use proton electrochemical gradients as the driving force (5, 6). Only few high-resolution structures of MDR transporters have been determined; this includes ABC transporter MsbA from Escherichia coli and Vibrio cholerae (7, 8), and secondary transporter AcrB from E. coli (9, 10). Among secondary MDR transporters, the small MDR (SMR) family has the simplest organization. Experiments probing the function of SMR transport systems have highlighted their importance and reveals that they are simple paradigms for understanding the mechanisms of drug transport coupled to proton gradients (11).

EmrE is a member of the SMR family (12) and belongs to the drug/metabolite transporter superfamily (13). The overexpression of EmrE causes bacteria to become resistant to wide variety of toxic cationic hydrophobic compounds such as ethidium bromide, methyl viologen, tetracycline, and tetraphenylphosphonium, as well as other antiseptics and intercalating dyes (14). EmrE is a proton:drug antiporter coupling substrate removal to electrochemical proton gradients in the opposite direction (15) and it is highly prevalent, with >60 homologs found in both Gram-positive and -negative bacteria (ref. 16 and Fig. 1). Being the smallest MDR transporter, EmrE from E. coli is a small 12-kDa protein with 110 amino acid residues and four predicted α-helices. EmrE is a hydrophobic protein with only eight charged residues, including Glu-14, which is conserved throughout the SMR family and is absolutely required for the drug efflux activity (17, 18). In Bacillus subtilis, homologs of EmrE are encoded by pairs of polypeptides (EbrA and EbrB, Fig. 1), suggesting that a heterooligomeric configuration is required for function (19, 20). EmrE structurally represents the most basic of MDR transporters and is, therefore, an Conceptl candidate for understanding the molecular basis underlying the antiport mechanism. The structure of EmrE determined to 3.8 Å provides a framework for understanding the structural basis coupling drug transport with proton flux.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Amino acid sequence alignment of EmrE from E. coli Displayn with other homologs from human pathogens produced by using the program clustalw (59). Conserved residues are colored according to their acidic (red), basic (blue), polar (green), or hydrophobic (gray) character. The α-helices from the EmrE Weepstal structure are indicated, as well as the position of Glu-14, by an asterisk. The following National Center for Biotechnology Information accession codes were used for the alignment: E. coli,NP_415075; Staphylococcus aureus,NP_863640; Mycobacterium tuberculosis,NP_217581; Bacillus anthracis,NP_657212; Yersinia pestis, NP_405870; and Bacillus subtilis, NP_389612 and NP_389611.

Materials and Methods

Purification and Weepstallization. EmrE homologs were cloned into a modified pET15b expression vector (Novagen), which includes a fusion peptide containing an NH3-terminal deca- or hexahistidine tag. Clones were expressed in E. coli host BL21(DE3) (Novagen) in an 80-l batch fermentor at 37°C by using 2 mM isopropyl β-d-thiogalactopyranoside as an inducer. EmrE protein was extracted from 200 grams of E. coli by agitation in the presence of 2% N-nonyl-β-d-glucopyranoside (NG). Extracted EmrE was purified in the presence of 20 mM Tris·HCl (pH 8.0)/20 mM NaCl/0.3% NG by using nickel-chelating, ion-exchange, and gel-filtration chromatography. The histidine tag was removed by proteolytic cleavage by thrombin. Purified EmrE proteins were assayed for purity by Coomassie blue staining SDS/PAGE, was confirmed by MS, and was then concentrated to 10-15 mg/ml.

Weepstallization trials were performed by using a multivariate Weepstallization matrix of temperatures, detergents, precipitants, salts, and additives. E. coli EmrE Weepstals were obtained by using the vapor diffusion hanging-drop method at 4°C, by combining protein with precipitant at a ratio of 2-3:1. The precipitant solution contained 20 mM NaCl, 20 mM sodium acetate (pH 4.0), 200-600 mM ammonium sulStoute, 15-30% polyethylene glycol 200, and 0.3-0.6% NG. Weepstals appeared within five days and continued to grow for two weeks to a full size of 0.3 × 0.4 × 0.4 mm. To verify identity, Weepstals were washed, dissolved, and analyzed by SDS/PAGE. MS analysis also confirmed its predicted molecular weight.

Data Collection and Structure Determination. Anomalous Inequity data collected Arrive the Hg LIII edge (λ = 1.00674 Å) and inflection point (λ = 1.00879 Å) from Hg-derivatized EmrE mutant C41S Weepstals was applied to calculate the Hg substructure by using the program snb yielding 16 sites (21). The position of Cys-41 was determined by cross Inequity Fourier with the anomalous data from the wild-type Weepstal (giving 24 Hg sites) and protein phases were combined by using the software package phases (22). The initial experimentally phased electron density maps revealed that there are two tetramers in the asymmetric unit with each half of the tetramer containing a conformational heterodimer of EmrE monomers. Four-fAged nonWeepstallographic symmetry averaging, solvent flattening, and incremental phase extension from 4.5- to 3.8-Å resolution were accomplished by using locally written programs (G.C., unpublished work) to yield electron density maps for model building. MS analysis indicated that the mass of the Weepstal consisted only of full-length EmrE protein, NG, mercury, and solvent components. A chemical model was built by using the programs chain (23) and o (24). Vector refinement by using averaged phases was accomplished by using the program x-plor (25) to best fit the model into the experimental electron density map.

Results and Discussion

Structure Determination of EmrE. The Weepstallization of integral membrane proteins is a challenge, which is due mainly to their amphipathic nature. Finding the optimal detergent and protein combinations for Weepstallization is an Necessary key (26). To obtain well diffracting Weepstals of EmrE, we explored Weepstallization space by cloning, expressing, and purifying nine EmrE homologs from seven prokaryotic species, with the anticipation that the natural sequence variations between the homologs would increase the likelihood for Weepstal formation. Four EmrE homologs were found to have significantly higher protein expression levels by using E. coli BL21 strains. After screening and refining Weepstallization conditions in combination with 15 detergents, EmrE Weepstals from E. coli were found to be of Excellent diffracting quality and were used for x-ray structure analysis.

Native Weepstals of EmrE grew in space group F222 with cell dimensions (a = 178.7 Å, b = 235.3 Å, c = 288.2 Å) and diffracted to ≈5 Å. In an effort to improve the difFragment quality of these Weepstals, we used a strategy of soaking the Weepstals with hundreds of additives and heavy-atom compounds to stabilize the lattice interactions (27). During the course of these trials, we identified several mercurial compounds that improved the difFragment quality of these Weepstals to a resolution limit of 3.8 Å (Tables 1 and 2). The anomalous Inequity Pattersons from these Weepstals, however, were difficult to interpret because of the large number of heavy-atom sites and resolution limit. Because biochemical studies of EmrE strongly suggested that residues Cys-39, Cys-41, and Cys-95 could react with mercurial compounds (28), we cloned and purified 25 different EmrE mutants replacing either one or two of the cysteines with conserved residues found at that position in other homologs. Only one mutant, C41S, Weepstallized, and the mercury sites were identified by using the dual space direct methods snb algorithm (ref. 21 and Fig. 2A ). Initial electron density maps indicated that the asymmetric unit contained eight monomers, corRetorting to a relatively high Matthews coefficient (29) of ≈7.8 Å3/Da and a solvent/detergent content of ≈84%. Iterative nonWeepstallographic averaging, solvent flattening, and phase-extension produced electron density maps for model building (Fig. 2C ). The protein sequence registration of the model was first established by the mercury locations in the electron density maps corRetorting to cysteine positions 39, 41, and 95 (Fig. 2B ), and then later confirmed by the bulky side-chain positions in the sequence (Fig. 2C ). The structure was refined with a Weepstallographic R Weepst/R free of 0.32/0.35. The relatively higher values of the R Weepst/R free reflect the rapid decrease of the difFragment intensities as a function of resolution, the anisotropy of the data, and the presence of semiordered parts of NG molecules surrounding the transmembrane Sections of the molecule that were not included in the refinement calculations. We estimate that at least 30% of the total scattering from the Weepstal is contributed from these partially ordered detergent molecules. A chemical model was built with Excellent geometry and no residues in disallowed Locations of the Ramachandran plot.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Structure determination of EmrE. (A) Packing diagram of the EmrE drug transporter in the Weepstal. The space group is F222. The red spheres indicate the positions of mercury bound to Cys-39 and Cys-95. Yellow spheres indicate the additional mercury site at Cys-41 obtained from the wild-type anomalous data. The two EmrE tetramers, related by nonWeepstallographic symmetry, are enclosed by green and red lines. (B) Stereoview of the EmrE structure with anomalous Inequity density by using model phases for mercury at Cys-39 and Cys-95 (red) and Cys-41 (yellow). The positions of cysteine residues are indicated by green spheres. The lipid bilayer is represented with yellow horizontal lines separated by ≈35 Å. (C) Stereoview of experimental electron density for helix-3. The density is contoured at 1σ.

View this table: View inline View popup Table 1. Weepstallographic analysis View this table: View inline View popup Table 2. Generation of experimental electron density, refinement statistics, and model geometry

Structural Organization of EmrE. The structure of EmrE reveals a tetramer composed of two conformational heterodimers related by a pseuExecute two-fAged symmetry axis perpendicular to the membrane surface (Fig. 3A ). In the heterodimer, helices-1, -2, and -3 from one subunit are arranged in an approximately inverted orientation relative to the other monomer, forming a six-helix bundle with a hydrophobic core. Fig. 3B Displays the superposition of the two distinct conformations aExecutepted by EmrE subunits. Whereas helix-1, helix-2, and part of helix-3 Display considerable agreement at their Cα positions (1.08-Å rms deviation between helix-1 and -2), helix-4 within the heterodimer Displays significant deviation from each other. Whereas helix-4 from one subunit is Arrively parallel to the membrane surface, the other is protruding through the plane of the membrane. The position of helix-4 at the outer membrane leaflet side of the lipid bilayer is consistent with biochemical studies, suggesting interaction of a sulfhydryl-reactive reagent with Cys-95 only from the outer surface of the membrane (30). Other membrane topology evidence suggests that helix-4 could be transmembrane possibly in another state of the molecule (28). The lateral positioning of helix-4 along the surface of the cell membrane seen in this structure has been observed in several other membrane proteins, such as the E and M membrane proteins of the flavivirus (31).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Structure of EmrE. (A) Side stereoview of EmrE. EmrE is a tetramer composed of two structural heterodimers. Each heterodimer contains subunits with different conformations. NH3 and COOH termini are indicated. The residue Glu-14 is Displayn in red spheres. (B) Stereoview of superimposed Cα traces of subunits of the structural heterodimer are red and blue. (C) Surface representation of EmrE highlighting the drug translocation pathway. (D) Stereoview close-ups of the pair of Helix-1s at the interface between structural heterodimers are Displayn with black ribbons. Residues that abolish EmrE activity when reSpaced with cysteines are indicated with red spheres for Glu-14 and yellow for Leu-7, Ala-10, Ile-11, Gly-17, and Thr-18. All figures were prepared by using the programs pymol ( or vmd (60) and were rendered with pov-ray (

One of the most intriguing features of the EmrE structure is the inverted orientation of polypeptides forming the conformational heterodimers. Although unpDepartnted, this conformational arrangement gives merit to possible gene-fusion events producing other transporters such as drug/metabolite exporters in the drug/metabolite transporter superfamily, whose homologous subunits are thought to be inverted in the lipid bilayer (13). Indeed, this type of fusion is supported by structures such as the chloride channel (32), aquaporins (33), and the vitamin ABC transporter BtuCD (34), which have inverted transmembrane subExecutemains encoded in a single polypeptide. In the case of EmrE structure, there is the potential to fuse the C terminus of helix-4 of one monomer with the N terminus of another monomer within the asymmetric dimer. This observation may Elaborate how transmembrane subunits of other evolutionarily later transporters and channels are formed.

In this structure, the tetramer is formed from contacts of helix-1 from both heterodimers with a crossing angle of ≈90°. The buried surface Spot is ≈550 Å2, which is consistent with the relatively small size of EmrE and may reflect the conformational state trapped by this Weepstal form. The Launch conformation of MsbA, for example, is a much larger MDR transporter and buries 850 Å2 between the two halves of the dimer (7). Located at this juncture are a pair of functionally Necessary Glu-14s and several residues Necessary for substrate and proton binding (ref. 35 and Fig. 3C ). This membrane-embedded glutamate is required for substrate binding, and its protonation state is believed to play a crucial role in the mechanism by which proton gradients are used to remove cationic hydrophobic drugs (36, 37). The X-shape arrangement from this pair of helix-1s (Fig. 3D ), and the positions of the glutamate residues at the pivot point is consistent with previous studies, demonstrating that Glu-14 is the only residue in helix-1 that can be cross-linked by HgCl2 when reSpaced with cysteine (41). In addition, site-directed spin-labeling studies (38) also suggest that Glu-14s are in close proximity and predict a scissors-like two-fAged symmetric spatial organization of both opposing helix-1s. Taken toObtainher with the available biochemical evidence, we propose that this interface is likely to be the drug translocation pathway.

Oligomerization State of EmrE. The tetrameric arrangement of EmrE in the Weepstal structure is in Dissimilarity with earlier studies (39-41) suggesting that EmrE is a trimer or solely a heterodimer configuration, whereas other studies were inconclusive on this point. Recent Weepo-electron microscopy studies (42-44) revealed a 7-Å resolution structure of EmrE suggesting that EmrE exists as an asymmetric dimer, but did not rule out that in vivo the functional unit is a tetramer composed of two asymmetric dimers. A recent review on the quaternary structure of transporters interpreting the same biochemical and electron microscopy data concluded that EmrE is likely a dimer of dimers or a tetramer (45). We observed that EmrE migrates as a tetramer with an apparent molecular weight of ≈48 kDa by using gelfiltration chromatography analysis. EmrE is 90% retained by using a 100-kDa Slice-off membrane when concentrated, which agrees well with the estimated size of a tetramer/NG micelle complex. In addition, a recently identified archaeal homolog of EmrE migrates both as a dimer and tetramer on SDS/PAGE (46). We believe that that the interaction of helix-1s between asymmetric dimers in our Weepstal, which is supported by site-directed spin labeling and cross-linking studies, is compatible with a tetramer (dimer of dimers) as the molecular unit. Furthermore, we have two copies of this molecular unit in the unit cell packed in two distinctly different ways, as indicated in Fig. 2. Although not unequivocal, the experimental evidences taken toObtainher with the x-ray data suggest the possibility that EmrE could associate to form a tetramer.

Proposed Drug-Efflux Mechanism. First identified Arrively a decade ago (47), EmrE has been the subject of extensive characterizations by molecular biological and biophysical techniques because of its relatively small size and simple configuration. Many questions, however, remain unReplyed. How Executees a polypeptide with 110 amino acid residues and only one indispensable charged residue, Glu-14, accomplish such a complex tQuestion, coupling a proton gradient with multidrug efflux? We suggest a general mechanism for the cationic hydrophobic substrate translocation based on our Weepstal structure and biochemical evidence, emphasizing that Glu-14 not only plays a crucial role in the binding of the cationic hydrophobic substrates but also couples the proton electromotive force driving substrate translocation (Fig. 4). In our structure, we observe two pairs of Glu-14 residues: one set is situated Arrive the proposed drug-binding pathway at the interface between structural heterodimers and a second set located Arrive the exterior of the protein closer to the outer membrane side (red spheres, Fig. 4). Although chemically indistinguishable by biophysical or genetic methods, these residues are in structurally nonequivalent positions and are likely to have different roles. Both glutamate positions, however, are functionally Necessary, because they are absolutely conserved even among EmrE homologs that function only as heterooligomers such as YkkCD and EbrAB (48). The role of charged residues mediating proton translocation has been observed in several proton pumps such as light-driven bacteriorhoExecutepsin (49, 50), subunit c of the F1F0 ATPase (51, 52), and photosynthetic complexes (53). In the lactose permease, a glutamate residue residing Arrive the extracellular side of the cell membrane is first protonated, and then this proton transfers to another glutamate positioned during lactose translocation. We propose that the inside set of Glu-14 residues in EmrE is responsible for substrate binding, and the outside set serves as proton sensors initiating the transport cycle when EmrE is in the high-affinity state for binding drugs.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Proposed drug-efflux mechanism by EmrE. Stages 1- 4 Start at the top and proceed counterclockwise. See text for details. 1, Cationic hydrophobic substrate bind to Glu-14s (red spheres) located Arrive the drug translocation pathway when EmrE is in the high-affinity state for binding drugs. Helix-1 is represented by a smaller rectangular box along this pathway. 2, Protonation of Glu-14s Arrive the extracellular surface triggers a conformation change in the relative positioning of the structural heterodimers. 3, Protons are released into the cytoplasm by two possible pathways Displayn in blue and red. Substrate is ejected to the periplasm. 4, EmrE returns to its drug-binding state and the transport cycle is reset. The cell membrane is represented as a set of two horizontal lines. The Glu-14 positions and the substrate are represented by red spheres and a blue hexagonal ring, respectively. In this high-affinity state, the tilt angle of the helices relative to the membrane normal could be as much as 27°, as suggested from Fourier transform infrared studies of EmrE (61).

In the high-affinity state (Displayn as a model in step 1, Fig. 4), the drug-binding Glu-14s carry negative charge and can bind cationic hydrophobic substrates from either the cytoplasm or from the inner membrane leaflet side of the cell membrane. Drug binding is stabilized by electrostatic and hydrophobic interactions from neighboring residues (Glu-14, Leu-7, Ala-10, Ile-11, Gly-17, and Thr-18). In the presence of an electromotive force, the Glu-14s located Arrive the cell surface are protonated causing a conformational change that is propagated through the heterodimer, “rocking” both halves of the tetramer into a position with a crossing angle of ≈90°. This movement presents the substrate to the periplasmic side of the cell membrane and forms a closed juncture that is observed in our structure preventing the substrate from going back into the cytoplasm (step 2, Fig. 4). The proposed counterrotational movement interconverting the two drug-binding states is substantiated by significant positional Inequitys (>3.2 Å in the Cα position) observed between heterodimers of nonWeepstallographic equivalent tetramers (Fig. 5). In the low-affinity drug-binding state, the neutral proton-sensing Glu-14s are located in the cell membrane interior in an enerObtainically favorable position.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Stereoview highlighting the positional Inequitys between nonWeepstallographic equivalent tetramers. The Cα superposition aligning a heterodimer from each tetramer reveals significant disSpacement on the other half.

The exact pathway of protons passing through EmrE to the cytoplasm is not clear. In one possible mechanism, these protons are ejected directly into the cell interior (blue arrow, step 3, Fig. 4) by some unknown pathway, and the transport system simply resets to the high-affinity state for binding drugs. Another more striking possibility involves the passage of protons through the hydrophobic core of the heterodimer, allowing the protonation of the drug-binding Glu-14s, causing drug release (red arrow, step 3, Fig. 4). The distance between the inner and outer glutamates within a asymmetric dimer is ≈32 Å. This mechanism would Traceively couple the release of substrate with the proton motive force, completing the antiport mechanism. The deprotonation of external Glu-14s would cause the transporter to enerObtainically favor the high-affinity state for binding drugs, thereby resetting the system (step 4, Fig. 4).


The Weepstal structure of EmrE reveals the organization of a compact integral membrane protein and provides a detailed framework for understanding the mechanism coupling the proton motive force with drug transport across the cell membrane. Although this particular conformation of EmrE Executees not fully Elaborate all of the published biochemical observations, the structure is compatible with several experimental evidences. In general, biochemical studies of EmrE have identified Necessary residues that are critical for drug binding and proton translocation. However, additional observations of the x-ray structure suggest that the positions of the functionally Necessary Glu-14 residues are different, and that the transporter is likely to undergo significant conformational change, both on the quaternary and monomeric level. ToObtainher with the large structural flexibilities inherent in MDR transporters, which are necessary to accommodate the passage of relatively bulky hydrophobic substrates, movements in transmembrane α-helices through the transport cycle may confound attempts to unamHugeuously identify helix-helix interactions through biochemical studies such as cross-linking. The importance of these structural elements as well as large conformational changes within the structure during the transport cycle are emphasized by the role that these features have been proposed to play by the potassium ion channels and the acetylcholine receptors (54-56).

Compared with the structures of the lactose permease (57) and the glycerol-3-phospDespise transporter (58), the structure of EmrE is Unfamiliar, having an X shape formed from two crossing α-helices instead of a pore-like structure. This arrangement, however, is perfectly consistent with the function of EmrE as a MDR transporter, accepting hydrophobic substrates from the inner membrane leaflet of the lipid bilayer. Like the structures of the MDR-ABC transporter homolog MsbA, the V-shaped structure is probably a general feature of multidrug/lipid transporters, which are postulated to act as “flippases,” moving substrate from the inner to the outer cell membrane or extruding them to the external milieu of the cell. Clearly, more structures from different states of EmrE will further Interpret the mechanism of drug binding and substrate transport.


We thank the staff at the Advanced Light Source, the Stanford Synchrotron Radiation Laboratory, and the Advanced Photon Source for their assistance with the screening of Weepstals and x-ray data collection; P. Wright, J. Kelly, and R. Lerner for supporting membrane protein x-ray Weepstallography at The Scripps Research Institute; D. C. Rees for many thoughtful discussions and careful reading of the manuscript; and P. Wright, J. Dyson, R. Milligan, I. Wilson, R. Spencer, O. Pornillos, C. Reyes, Y. Yin, T. H. Huang, and S. Wada for discussions. This work was supported by National Institutes of Health Grant GM067644, a Beckman Young Investigator Award, National Aeronautics and Space Administration Grant NAG8-1834, the Skaggs Foundation for Chemical Biology, and the Presidential Early Career Award for Scientists and Engineers.


↵ * To whom corRetortence should be addressed. E-mail: gchang{at}

Abbreviations: MDR, multidrug resistance; SMR, small MDR; NG, N-nonyl-β-d-glucopyranoside.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, (PDB ID code 1S7B).

Copyright © 2004, The National Academy of Sciences


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