Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.https://doi.org/10.7554/eLife.07380.001
Gram-negative bacteria can cause serious diseases in humans, such as cholera and bacterial meningitis. These bacteria are surrounded by two membranes: an inner membrane and an outer membrane. Proteins called secretins are components of several large molecular complexes that are embedded within the outer membrane. Some secretin-containing complexes form pores in the bacterial membranes and allow molecules to pass in or out of the cell.
Some secretins also form part of the machinery that allow Gram-negative bacteria to grow fibre-like structures called type IV pili. These pili help bacteria that cause infections to move and stick to host cells, where they can also trigger massive changes in the host cells' architecture. Multiple copies of a secretin protein called PilQ form a channel in the outer membrane of the bacteria that allows a type IV pilus to grow out of the surface of the cell. The pilus can then hook the bacteria onto surfaces and other cells. There is evidence to suggest the type IV pilus machinery is involved in the uptake of DNA from other bacteria, an important but poorly understood process that has contributed to the spread of multi-drug resistance.
Now, Gold et al. have used a cutting-edge technique called ‘electron cryo-tomography’ to analyse the three-dimensional structure of the machinery that builds the type IV pili in the membranes of a bacterium called Thermus thermophilus. This analysis revealed that, similar to many other channel complexes, the PilQ channel can be ‘open’ or ‘closed’. When pili are absent, the channel is closed, but the channel opens when pili are present. Further analysis also revealed the structure of an assembled pilus.
Next, Gold et al. studied the open state of the type IV pilus in more detail and observed that a region of each of the PilQ proteins moves a considerable distance to make way for the pilus to enter the central pore. These results will pave the way for future studies of type IV pili and other secretin-containing complexes and underpin efforts to investigate new drug targets to combat bacterial infections.https://doi.org/10.7554/eLife.07380.002
Secretins form multimeric pores through the outer membrane of Gram-negative bacteria (Averhoff, 2009; Korotkov et al., 2011; Burkhardt et al., 2012). They are the central secretion conduits for proteins and virulence factors in type II and III secretion systems (T2SS/T3SS) and are essential for extrusion of type IV pili (T4P) (Martin et al., 1993) and transport of some bacteriophages (Korotkov et al., 2011). In addition, secretins are key components of DNA transport systems, which mediate uptake of free DNA from the environment, referred to as natural transformation (Schwarzenlander et al., 2009). The ability to take up DNA is one of the major mechanisms of horizontal gene transfer (Domingues et al., 2012) and enables organisms to adapt rapidly to changing environments (Averhoff, 2009). This process is also fundamental for adaptation of pathogenic bacteria to human hosts and the acquisition of multi-drug resistance (Domingues et al., 2012).
In many Gram-negative bacteria, such as Thermus, and also in many major human pathogens such as Neisseria, Pseudomonas, and Vibrio, DNA uptake is linked to the T4P machinery (Wolfgang et al., 1998; Graupner et al., 2000; Seitz and Blokesch, 2013) (Figure 1). To investigate the structure and function of this system, we chose the thermophilic bacterium Thermus thermophilus HB27, which exhibits the highest transformation rates known to date (Koyama et al., 1986), and due to the thermostability of its proteins is a convenient model for structural studies.
Pili are several micron-long flexible filaments (Craig and Li, 2008) that can generate forces of over 100 pN (Maier et al., 2004). T4P are grouped together in a class based on the production and secretion of the major pilin protein PilA4 (Thermus nomenclature), thousands of copies of which form the helical pilus (Craig et al., 2004; Schwarzenlander et al., 2009). The T4P is the only known bacterial pilus that can be retracted rapidly (Maier et al., 2004) to enable motility and adherence (Merz et al., 2000), major contributors to bacterial virulence (Hahn, 1997). Assembly and disassembly of the pilus is driven by the AAA-ATPases (ATPases associated with diverse cellular activities) PilF (extension) and PilT1/PilT2 (retraction) (Salzer et al., 2014b). It has been suggested that mature PilA4 assembles into pili extending from the inner membrane by action of PilF (Collins et al., 2013; Salzer et al., 2014b). The outer membrane channel of the T4P machinery is formed by the dodacemeric ∼1 MDa secretin complex PilQ (Burkhardt et al., 2011) (Figure 1). Other proteins, in particular PilM, PilN, and PilO, are hypothesized to be a central part of the pilus assembly platform and may couple the cytoplasmic and periplasmic sides of the T4P machinery (Karuppiah et al., 2013). Some proteins of the complex have been implicated to play a dual role in both pilus assembly and natural competence (Friedrich et al., 2002; Averhoff and Friedrich, 2003; Friedrich et al., 2003; Rumszauer et al., 2006). Recent results indicate that T4P themselves are not directly involved in DNA uptake (Burkhardt et al., 2012; Salzer et al., 2014a).
T4P are essential for pathogenesis by mediating adhesion, biofilm formation, and twitching motility (Burrows, 2012). Thus, both secretins and T4P play important roles in virulence of different pathogenic bacteria, which has fostered their use as new targets for drug development (Baron, 2010). To date, there is no information on the in situ structure of either the T4P machinery or DNA translocator. Determining structures of T4P complexes in whole bacterial cells is therefore of paramount importance and will enable further study of bacterial resistance and disease. Electron cryo-tomography (cryoET) has the unique ability to determine protein structures in cells at molecular resolution. We have applied cryoET to whole T. thermophilus HB27 cells, in order to visualize the T4P machinery in situ. We determine the helical structure of the pilus and find that the secretin complex PilQ is a central dynamic component of this system. CryoET and subtomogram averaging of the T4P machinery with and without pili reveal a ∼30 Å conformational change as the gates in the complex open.
T. thermophilus has an unusual cell architecture with deep surface clefts, formed by invaginations of the outer membrane (Figure 2A). By cryoET, these clefts are seen to be constrictions that run around the cell body (Figure 2D). Distal to the most polar outer membrane ring, numerous fibrous and straight pili extend from the cell (Figure 2A–D). The pili are clearly associated with large protein complexes crossing the ∼70 nm periplasm (Figure 2B,C). This distribution is in line with previous fluorescence and electron microscopy data, which demonstrate the polar localization of PilQ (Seitz and Blokesch, 2013) and pili (Salzer et al., 2014c).
The periplasm of T. thermophilus cells is extraordinarily wide (Quintela et al., 1995; Castan et al., 2002) and too dense to select subvolumes for subtomogram averaging reliably. Therefore, cells were treated with 100 mM ethylenediaminetetraacetic acid (EDTA) and pipetted in order to render the outer membrane leaky (Caston et al., 1988). This had the desired effect of depleting the periplasm of most small proteins. Sample preparation by this method also removed the pilus from the complex, the empty T4P machinery was nonetheless still clearly visible (Figure 3A,B). We determined by subtomogram averaging the structure of the entire complex and found features distinct from those of the T2SS and T3SS secretins (Marlovits et al., 2006; Hodgkinson et al., 2009; Reichow et al., 2010). The resolution obtained by averaging ∼4000 particle subvolumes was ∼35 Å (Figure 3—figure supplement 1), most likely limited by the inherent flexibility of the complex (Burkhardt et al., 2011) and the difficulty of correcting precisely the contrast transfer function (CTF) for thick specimens.
Subtomogram average maps show the central protein channel (∼35 nm long and ∼15 nm wide) made up of several ring-shaped domains inserted into the outer membrane (Figure 3C, left panel). We compared a 2D projection of the channel part of the subtomogram average with projections of purified, negatively stained PilQ only (Burkhardt et al., 2011) (Figure 3C, central and right panels). PilQ was seen to consist of a C-terminal trapezoid ‘cone structure’ with staggered rings in the N-terminal domain (Burkhardt et al., 2011), in excellent agreement with our in situ structure. Moreover, the new cryoET structure shows additional protein densities extending from the putative N0 domain of PilQ through the peptidoglycan layer (P1 and P2) to the cytoplasmic membrane (C1) (Figure 3D). Candidate proteins include PilW, which is associated with the inner and outer membranes and is essential for the outer membrane localization of PilQ (Rumszauer et al., 2006), and PilO/PilN heterodimers that could connect PilQ to the ATPases by PilM in the cytoplasm (Karuppiah and Derrick, 2011; Karuppiah et al., 2013) (Figure 1). These proteins are most likely connected to one another by flexible domains that are not well contrasted in the subtomogram average. A longitudinal slice through the complex reveals that PilQ has two gates, which are closed in the absence of a pilus (Figure 3C, left panel and Figure 3D, right panel). Gate 1 is formed by the ‘cone’ in the outer membrane and gate 2 by the N1 domain at the base of PilQ, enclosing an empty periplasmic vestibule (Figure 3D, right panel). The C-terminal ‘cone’ has been shown to form a sodium dodecyl sulfate (SDS)-stable sub-domain (Burkhardt et al., 2011), thus, it is plausible that gate 1 is responsible for maintaining the integrity of the cell membrane in the closed state. A second gate formed by the N-terminal domains has not been observed in other secretins (Korotkov et al., 2011). The N-terminus of PilQ forms an unusual βββαββ fold, different from the conserved ring-building βαββα folds (Burkhardt et al., 2012). Thus, we hypothesize that this motif may form part of gate 2.
To determine the structure of the T4P machinery in the open state, the pipetting step during sample preparation was omitted (‘Materials and methods’), which reduced the shearing forces and kept the assembled pilus intact (Figure 4A,B). Close inspection of the pilus shows a periodic structure (Figure 4C), which was suitable for subtomogram averaging. Since the ice surrounding the pili was thin (∼100 nm), it was possible to apply CTF-correction. The structure of the pilus was determined at ∼32 Å resolution by averaging 740 subvolumes (Figure 3—figure supplement 1). Power spectra calculated from a single tomographic slice or from the subtomogram average revealed a repeat distance of ∼4.9 nm (Figure 4C, lower panels). The T. thermophilus pilus forms a right-handed helix of ∼3 nm diameter (Figure 4D), which is different from the previously determined electron cryo-microscopy structure of the isolated Neisseria gonorrhoeae T4P (Craig et al., 2006). Diameters of pili can vary considerably and pilin proteins have limited sequence similarity (Craig and Li, 2008), which likely accounts for this difference. The result may prompt a reassessment of the functional roles of different T4P in cells. Due to the more challenging sample preparation method, the structure of the open T4P machinery with the pilus extended was determined from only ∼300 particles at ∼45 Å resolution (Figure 3—figure supplement 1). This resolution is sufficient to reveal the central protein channel complex with the ∼3 nm pilus protein density in the centre (Figure 4E). An extensive conformational change is evident that shifts PilQ domains N0–N3 away from N4/N5 by ∼30 Å towards the cytoplasmic membrane. This change opens the periplasmic vestibule to make way for the pilus (Figures 4E, 5A,B and Video 1). The shape and dimensions of the pore change from a tapered form ranging in width from ∼4 nm (at N5) to ∼8 nm (at N2/N3) in the closed state (Figure 3C), to a roughly constant 7 nm-wide channel in the open state (Figure 4E). In comparison, the closed state of Neisseria meningitidis PilQ is ∼9 nm wide tapering to a point (Collins et al., 2004), which would be sufficient to accommodate the wider Neisseria pilus (Collins et al., 2003; Craig et al., 2006) after a comparably large conformational change. Additional conformational changes and shifts also occur in protein densities P1, P2, and C1 (Figure 5A). In the open state, we observe an extra protein density in the cytoplasm (Figure 4E, yellow arrowheads and Video 1), which we speculate could be PilF, linked to the inner membrane platform via PilM (Karuppiah and Derrick, 2011) (Figure 1). Homologous proteins have been shown to interact with the cytoplasmic ATPases, stimulating their activity (Lu et al., 2013, 2014). Because EDTA treatment caused a depletion of periplasmic protein, we cannot exclude the possibility that some proteins may have been removed from the T4P machinery. However, we also averaged complexes from a tomogram of the two cells shown in Figure 2, which contained both open (with pili) and closed (without pili) complexes and where the periplasmic protein density was considerably higher. These subtomogram averages (Figure 5—figure supplement 1) show clearly that the same large conformational changes occur in the T4P machinery, irrespective of the degree of periplasmic protein depletion, and hence of the effect of EDTA.
Docking the structures of the closed complex back into the tomographic volume reveals their three-dimensional distribution in the cell (Figure 5C,D and Video 2). We find that the T4P machinery tends to be tightly packed, with an inter-particle distance of 30–40 nm (Figure 5E). When the pili were depleted by pipetting, each cell contained on average 33 ± 19 closed complexes. However, T. thermophilus assembles ∼6 pili per cell (Salzer et al., 2014a, 2015), suggesting that ∼80% of the complexes are not involved in pilus formation under standard growth conditions (Salzer et al., 2014c). We speculate that these idle complexes may form a second class of transporter, which may be active in DNA uptake or protein secretion.
Taken together, our results demonstrate that the DNA translocator protein PilQ forms a dynamic central component of the T4P machinery in T. thermophilus. The length of the complex across the wide periplasm may be an adaptation to the thermophilic environment that Thermus thrives in. However, core components of the T4P machinery are conserved in bacteria (Nudleman and Kaiser, 2004), and thus, we speculate that the overall architecture may be similar. Our findings will enable further structure-function studies of the proteins that comprise this elaborate and important macromolecular machine.
T. thermophilus HB27 was grown in TM+ medium (8 g/l tryptone, 4 g/l yeast extract, 3 g/l NaCl, 0.6 mM MgCl2 0.17 CaCl2) (Oshima and Imahori, 1971). Cells from a 24-hr pre-culture were transferred onto TM+ plates (containing 2% [wt/vol] agar) and incubated under humid conditions for 48 hr at 68°C.
To determine the structure of the closed complex, cubes of agar with growing T. thermophilus cells were cut out and placed into buffer containing 20 mM Tris pH 7.4, 100 mM EDTA and gently agitated for 1 hr at room temperature. Samples were mixed 1:1 with 10 nm protein A-gold (Aurion, Wageningen, The Netherlands) as fiducial markers and applied to glow-discharged R2/2 Cu 300 mesh holey carbon-coated support grids (Quantifoil, Jena, Germany) by gentle pipetting. For the structure of the open complex, cells were treated with EDTA as above, then protein A-gold was added and grids dipped into the solution without the pipetting step. Grids were blotted for ∼4 s in a humidified atmosphere and plunge-frozen in liquid ethane in a home-made device. Grids were maintained under liquid nitrogen and transferred into the electron microscope at liquid nitrogen temperature.
Tomograms were typically collected from +60° to −60° at tilt steps of 2° and 5–7 μm underfocus, using either a Tecnai Polara or Titan Krios microscope (FEI, Hillsboro, USA), both equipped with field-emission guns operating at 300 kV and Quantum energy filters (Gatan, Pleasanton, USA) operated at a slit width of 20 eV. Both instruments were fitted with K2 Summit direct electron detector cameras (Gatan, Pleasanton, USA). Dose fractionated data (3–5 frames per projection image) were collected using Digital Micrograph (Gatan, Pleasanton, USA) at a nominal magnification of 34,000× (corresponding to a pixel size of 0.66 nm) in the Polara or at 33,000× (corresponding to a pixel size of 0.42 nm) in the Krios. The total dose per tomogram was ∼140e−/Å2. Tomograms were aligned using gold fiducial markers and volumes reconstructed by weighted back-projection using the IMOD software (Boulder Laboratory, Boulder, USA) (Kremer et al., 1996). Contrast was enhanced by non-linear anisotropic diffusion (NAD) filtering in IMOD (Frangakis and Hegerl, 2001). Segmentation was performed using AMIRA (FEI, Hillsboro, USA).
Data collected at 34,000× and 8 μm underfocus on the Tecnai Polara were used to calculate the subtomogram averages shown in Figure 5—figure supplement 1. Subtomogram averages of the T4P machinery shown in all other figures were calculated from data collected at 33,000× and 7 μm underfocus on the Titan Krios microscope. Coordinates corresponding to the outer membrane and inner membrane domains of the complex were marked manually in IMOD (Kremer et al., 1996). Subvolumes from twice-binned tomograms were then extracted from NAD filtered (Frangakis and Hegerl, 2001) data and an initial alignment and averaging performed in SPIDER (Frank et al., 1996). This average was used as a reference for alignment and refinement using PEET (Nicastro et al., 2006). We have previously shown that T. thermophilus PilQ is a dodecamer by biochemical analysis (Burkhardt et al., 2011), which is supported by single-particle electron microscopy of N. meningitidis PilQ (Collins et al., 2003, 2004). Therefore, 12-fold symmetry was applied to the core complex by 30° (360°/12 subunits) rotation of each subvolume prior to the alignment search. The final averages were obtained from 3984 particles for the closed complex and 312 particles for the open complex, using a mask drawn around PilQ. Any duplicates due to oversampling were removed in PEET (Nicastro et al., 2006). Due to the larger sample size, data for the closed complex were replaced by unfiltered tomograms. For 2D comparisons between states (Figure 3C, left panel and Video 1), the NAD filtered version with low-contrast noise removed is shown. For the pilus, 740 subvolumes of ∼2 nm length were selected in IMOD (Kremer et al., 1996), from unbinned CTF-corrected tomograms collected at 5 μm underfocus on the Titan Krios microscope. Subvolumes were aligned and averaged with a cylindrical mask and any duplicates due to oversampling were removed in PEET (Nicastro et al., 2006). Resolution estimates were obtained using conventional ‘even/odd’ Fourier shell correlation (FSC), applying the 0.5 FSC criterion. A mask was drawn around the protein to exclude membrane and peptidoglycan from this estimate. The averages in Figure 5—figure supplement 1 were smoothed by Gaussian filtering in UCSF Chimera, which was used to draw all surface views and remove low-contrast background noise using the ‘hide dust’ tool (Pettersen et al., 2004). The morph shown in Video 1 was produced in ImageJ (Schneider et al., 2012). All subtomogram averages were uploaded to the EMDataBank (http://www.emdatabank.org) with ID codes 3021 (closed state, filtered), 3022 (closed state, unfiltered), 3023 (open state, filtered) and 3024 (pilus).
The distance between PilQ complexes was determined with a MATLAB (Mathworks, California, USA) script (Gold et al., 2014). The centroid coordinates of complexes selected for subtomogram averaging were loaded into MATLAB and the distances calculated in an iterative for for-loop according to Pythagoras' theorem. This was performed for 332 closed PilQ complexes, combined from 9 different cells. Averaged histograms were calculated to depict the mean frequency of occurrence for each minimal distance. To account for the different numbers of PilQ complexes in each data set, the mean frequency was calculated as a percentage.
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Stephen C HarrisonReviewing Editor; Harvard Medical School, Howard Hughes Medical Institute, United States
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Thank you for sending your work entitled “Structure of a type IV pilus machinery in the open and closed state” for consideration at eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editor and another Jan Löwe (peer reviewer).
The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.
The short report titled “Structure of a type IV pilus machinery in the open and closed state” by Gold et al. describes the structure of a type IV secretion pore in two different states, bound and unbound to the pilus using cryo-electron tomography and sub-tomogram averaging. The message of the manuscript is straightforward. Although true molecular interpretation of the reported results will have to await studies at much higher resolutions, it is valuable to get an initial overview of what is happening in cells. The work presented is a very good example of what is currently possible.
A general concern of the reviewers was that only someone with considerable background in this field can follow the description and relate it to what is known from high resolution studies of components—e.g., the crystallographic studies the assembly platform (Karuppiah, et al.), secretion complex (Burckhardt, et al.), and fiber (Karuppiah, et al. and Craig, et al.). As the authors point out, the complex depicted in Figure 3C closely resembles that seen by Burckhardt, et al., but with some interesting structural differences. We therefore request a revised version that addresses the following points and that will allow a structural biologist, who does not know about bacterial pili, to understand the key points.
A) It would be helpful to use consistent terminology when introducing complexes and concepts. For example, the authors first introduce “secretins,” followed by reference to “the T4P system” and “the T4P machinery.” The molecular constituents of the T4P system/machinery/complex should be clearly delineated. Does T4P refer to the PilQ secretin complex alone (please see the Introduction), or is it meant to include both PilQ and PilMNO? Are PilMNO couplers, or parts of the complex? A cartoon of the model currently supported by the literature would be helpful.
B) Is the multi-ring structure, N0-N5, thought to be composed only of domains of the single protein (PilQ) or are there other components specific to particular rings. That is, do the authors think that the “gate closure” is a conformational change in PilQ itself or rather a redistribution of another protein?
C) Related to B). A major finding of the paper is a substantial conformational transition between the open and closed T4P machinery. (i) Since there are no atomic structures fitted into the cryoEM density presented, the importance of gate 2 in recognising the growing pilus is speculative. The entire paragraph should be reworded with more caution. (ii) The closed data set comes from EDTA-treated, pipetted, pilus-deficient cells, while the open set comes from non-pipetted, pilus-positive complexes. Comparison of the average number of pili to the average number of complexes suggests, as the authors write, that 80% of complexes are not associated with pili. Do these pilus-free complexes resemble the “closed” complexes seen in the pipetted, EDTA-treated cells?
D) To obtain the open state, the authors use strong EDTA treatment of cells to deplete the periplasm for the open state. This is an unnatural state for the pore proteins. Without this treatment, the signal to noise ratio is low in the tomograms, but the pore proteins can nonetheless be identified even without it (Figure 2B-C). Did the authors attempt to carry out subtomogram averaging from these native data? What was the result? It would be interesting to know the result, even just from an image processing perspective.
E) Related to D). The authors should introduce a caveat in the Results section that some molecules associated with the secretion pore may have been removed by the EDTA treatment and not make such strong points about “in situ” structures: the periplasm has been shrunk and emptied, at least partially.
F) The authors comment that T. thermophilus was chosen due to its high transformation rate and convenient thermostability (Introduction). Could the authors comment on how their in situ findings might be generalized to other bacterial species that do not have unusually deep surface clefts or an “extraordinarily wide” periplasm (as stated in the Results and Discussion section)? Is it possible that these characteristics are responsible for exaggerated structural dynamics that more readily facilitate DNA uptake and thus the higher transformation rate?
G) Some slightly more technical points:
i) The IMOD reconstruction showing the pilus does not show strong features. It is hard to see repeats in Figure 4C. Would it be possible to see a Fourier transform with a peak? The pili emanating from cells in Figure 2 are fairly long. Did the authors try to get 2D averages from the cryoEM images? If there is a repeat, then it should be easily visible from a few images. The issue is important because there are such strong differences from the pili of N. gonorrhoeae. In Neisseria, would one expect the T4P channel to be that much wider? Does it use other proteins or just more of them with more than 12-fold symmetry? Independent alignment and averaging of the present data should be tried, perhaps using the 0.143 FSC criterion.
ii) Is there any independent verification that T4P channels have 12-fold symmetry? The authors make heavy use of the symmetry, but it seems from the MS that they are relying on someone else's result.
iii) How confident can we be of the correspondence between the rings in the image from subtomogram averaging and the rings in the projection from negative staining of PilQ (Figure 3C)? The spacings seem rather different. Could other components contribute to the rings in the intact sheath?https://doi.org/10.7554/eLife.07380.012
- Vicki AM Gold
- Werner Kühlbrandt
- Ralf Salzer
- Beate Averhoff
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Bertram Daum for helpful comments on this manuscript, Deryck Mills for excellent maintenance of the EM facility, Andreas Walter for the MATLAB script, and Paolo Lastrico (Graphics Department, MPI of Biophysics, Frankfurt MPI) for assistance with Figure 1. This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (AV 9/6-1).
- Stephen C Harrison, Reviewing Editor, Harvard Medical School, Howard Hughes Medical Institute, United States
© 2015, Gold et al
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