Three-Dimensional Structure of the Neisseria meningitidis Secretin PilQ Determined from Negative-Stain Transmission Electron Microscopy

The PilQ secretin from the pathogenic bacterium Neisseria meningitidisis an integral outer membrane protein complex which plays acrucial role in the biogenesis of type IV pili. We present herethe first three-dimensional structure of this type of secretinat 2.5-nm resolution, obtained by single-particle averagingmethods applied to the purified protein complex visualized ina negative stain. In projection, the PilQ complex is circular,with a donut-like appearance. When viewed from the side it hasa rounded, conical profile. The complex was demonstrated tohave 12-fold rotational symmetry, and this property was usedto improve the quality of the density map by symmetry averaging.The dominant feature of the structure is a cavity, 10 nm deep,within the center of the molecule. The cavity is funnel-shapedin cross section, measures 6.5 nm in diameter at the top ofthe complex, and tapers to a closed point, effectively blockingformation of a continuous pore through the PilQ complex. Theseresults suggest that the complex would have to undergo a conformationalchange in order to accommodate an assembled pilus fiber of diameter6.5 nm running through the outer membrane.

INTRODUCTION

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Introduction

Bacteria express a variety of surface-located adhesive moleculesto achieve the necessary cell-cell contact required for initialcontact with a human host (20, 38). During tissue colonization,Neisseria meningitidis (the meningococcus) and many other gram-negativebacteria produce long adhesive fibers, termed type IV pili (Tfp),which emanate from the cell surface and mediate attachment viaspecific epithelial tissue receptors (17, 21, 47). In addition,Tfp have roles in diverse bacterial processes, including bacterialautoagglutination (48), variation of target tissue specificity(22), and natural competence for DNA uptake by transformation(15, 53). Tfp are responsive organelles, and the retractionof these fibers is directly responsible for bacterial twitchingmotility along solid and mucosal surfaces (28, 30). Assemblyof pilin subunits into Tfp is a multistage process and is controlledby between 14 and 30 genes (1, 46, 52). Many of the componentsof the Tfp biogenesis machinery are homologous to those engagedin the general type II secretory pathway (GSP) (33, 39).

The PilQ complex is an antigenically conserved, highly abundantouter membrane protein and is a member of the GSP secretin superfamily(39), members of which are involved in the terminal translocationof proteins and macromolecules across the outer membrane (2,50). The PilQ complex is critical for the surface expressionof Tfp, and meningococcal mutants lacking or expressing defectivePilQ are devoid of pili and pilus-associated phenotypes (8,50). A total of 200 to 300 conserved C-terminal residues inthe transmembrane region of the PilQ monomer exhibit the highestsequence homology to other secretins and are essential for oligomerizationinto the functional complex (18, 32, 50), although PilQ alsorequires a lipoprotein, PilP, for stable oligomerization (7).Functional PilQ in N. meningitidis forms a large ( $~$ 960-kDa) homododecamericcomplex (4) that is very stable and resistant to sodium dodecylsulfate (SDS) and heat treatment (50). The naturally high expressionlevel of a single secretin in meningococci favors this systemfor secretin characterization. Secretins from a wide range ofbacteria have been purified and observed by electron microscopy(EM) (2-6, 9, 23, 26, 31, 34, 44, 45, 49), and all of thesestudies have reported a ring-shaped complex composed of 6 to14 subunits. In each case, the ring enclosed a central cavityof various dimensions, and it is widely assumed that the secretedsubstrate passes through the body of this channel. An EM studyon intermediates in type I pilus biogenesis has provided evidencethat the FimD secretin does indeed act as a conduit for typeI pili (42).

Although these EM observations have been useful, structure-functionstudies of individual secretin complexes have been hamperedby a lack of three-dimensional (3-D) detail. Determination ofthe 3-D structure of the PilQ complex would provide novel andbiologically pertinent information on the nature of the centralcavity or pore: is a continuous channel formed through the PilQcomplex and, if so, is it of appropriate dimensions to accommodatepreassembled Tfp? One approach to obtain this type of 3-D structuralinformation for particles found predominantly in a single orientationis to use single-particle averaging (SPA) in combination withvolume reconstruction methods (12, 40). The only 3-D secretinstructures determined thus far are PulD from Klebsiella oxytoca(31) and the pIV protein from filamentous bacteriophage (34).

We report here the 3-D structure of the PilQ complex from N.meningitidis, obtained from single particles visualized in anegative stain. We find that the secretin forms a noncontinuouspore 6.5 nm in diameter that tapers substantially toward oneend. The results suggest a "gated pore" mechanism whereby thesecretin does not merely function as a passive channel or sheathbut plays an intimate part in regulating the extrusion or retractionof the nascent Tfp.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains. Meningococcal strain M1080 was grown overnight on 5% blood agarin an atmosphere containing 10% CO₂ before being harvested.The plasmid pMF121, generously provided by Mathias Frosch (16),was used to generate a capsule negative mutant expressing truncatedlipooligosaccharide of strain N. meningitidis strain M1080 bytransformation with erythromycin selection.

Purification of meningococcal PilQ. Meningococcal cells were disrupted in a French press in thepresence of the protease inhibitors phenylmethylsulfonyl fluoride,leupeptin, pepstatin A, and 2-mercaptoethanol (Sigma). The cellenvelope fraction was extracted in 4% (wt/vol) deoxycholatein buffer A (100 mM NaCl, 20 mM KH₂PO₄, 50 mM KCl, 5 mM EDTA,10 mM Tris; pH 7.5) and pelleted. The pellet was subsequentlydissolved in a buffer containing 4% (wt/vol) Zwittergent 3-10and 20% (vol/vol) glycerol and then dialyzed in buffer A with0.1% (wt/vol) Zwittergent 3-10. After concentration and sizeexclusion in Microsep300 columns (Filtron), the preparationwas loaded onto a 15 to 40% (wt/vol) continuous sucrose gradientand ultracentrifuged at 106,000 x g (4°C) for 24 h in aBeckman SW41 rotor. PilQ-containing fractions were selectedby immunoblotting and subjected to prolonged dialysis in bufferA with 0.1% (wt/vol) Zwittergent 3-10. PilQ complexes were concentratedagain in Microsep300 columns before use.

Sample purity. The presence of PilQ in whole-cell lysates and fractions duringpurification was detected by immunoblotting with rabbit polyclonalantibodies raised against purified N. meningitidis PilQ complexfrom strain M1080 at a dilution of 1:4,000 (50). Conditionsto verify PilQ sample purity by (i) SDS-PAGE, (ii) immunoblottingwith antibodies to PilE, PilP, PilC, and lipopolysaccharide,and (iii) antigen detection have been previously described (4,50, 51).

EM sample preparation and image scanning. Samples of PilQ were adsorbed to freshly glow discharged carboncoated grids (no. 400) as previously described (4), with thefollowing minor changes. The carbon sides of the grids wereplaced on the surface of droplets in the following sequence:20-µl PilQ sample droplets (100 µg/ml) for 10 min,followed by a 10-s wash in buffer (10 mM Tris-HCl [pH 7.5],100 mM NaCl, 5 mM EDTA), and finally a 10-min incubation infresh 2% (wt/vol) uranyl acetate. Table 1 shows all additionalinformation pertinent to transmission EM low-dose data collectionand image processing.

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TABLE 1. EM and image analysis information

Image processing. 3-D volumes of Zwittergent-solubilized PilQ were calculatedby the random conical reconstruction method (12, 13, 37, 40),implemented by using the SPIDER and WEB image processing softwaresuite (14) (www.wadsworth.org/spider_doc/spider/docs/master.html).Micrograph pairs of negatively stained PilQ complexes were selectedand scanned as described in Table 1. Deeply stained individualcomplex pairs presenting a clear projection (shown by arrowson Fig. 1A) were extracted into 26-nm² windows from the untiltedand tilted micrographs, and the individual CTF correction andfocus gradient were evaluated for each tilt pair (12).

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FIG. 1. PilQ particles visualized under negative stain. (A) Tilt pairs of PilQ particles at 0° and 40°, negatively stained with 2% uranyl acetate and recorded under low-dose conditions. Arrows indicate particles presenting the face-on donut view. Scale bar, 30 nm. (B) Montage of particles presenting putative side-on views of PilQ. Measurements of individual particles found widths and heights in the range of 15 to 16.5 nm and 18 to 20 nm, respectively. Scale bar, 15 nm. (C) 2-D projection maps of the two major class averages (n = 200 and 303) of particles selected for volume back projection. Maps are low-pass filtered to 2 nm and contoured at 20 levels. Scale bar, 10 nm.

The untilted data set was rotationally and translationally alignedby using previously described strategies (4) that resulted intwo closely related groups (n = 200 and 303). The alignmentfor each group produced was judged to be accurate by using acombination of criteria. (i) Independent alignment algorithmsproduced the same classification groupings. (ii) Correspondenceanalysis and hierarchical ascendant classification showed thatall groupings were highly related, with minimal variation atthresholds of $<=$ 0.01. (iii) Similarity to previous alignmentsproduced from a 2-D data set (n = 650) of SDS-solubilized PilQ(4) was the last of the criteria. The calculated shifts androtations were then applied to the data set after low-pass filteringto 2 nm, ramping, and contrast enhancement. Correspondence analysis(24, 25) and hierarchical clustering (37), with complete linkageas a merging criterion, were used to separate out the two majorgroups. Each of the two major volumes produced was individuallyrefined six times by optimizing the original volume by usingan angular refinement step. Back-projections calculated fromthe initial 3-D volume were used to translationally realignthe original tilted particles, and the resulting realigned particleswere then used to calculate a new volume. The two refined volumeswere then merged after determining the correct Euler anglesfor combination producing a final reconstruction, including503 particles prior to symmetry averaging. The final volumeproduced was judged to be a reasonable size for a PilQ dodecamer,assuming a packing density of 0.7 g/cm³; volumes calculatedat thresholds of 1 and 2 ${sigma}$ above the mean density could accommodatemolecular masses of 1,000 and 840 kDa, respectively. In orderto confirm that the volumes calculated from PilQ were presentedin a single orientation, higher clustering thresholds were usedto produce more class groups, each containing fewer particles.All of the volumes produced in this way had the same asymmetryor tapering features along the complex height. If PilQ volumeswere calculated from ring projections related by 180°, thecomplex would not show tapering consistently localized at oneend.

Self-orientation rotational and rotational power spectrum analyseson the final volume confirmed the 12-fold symmetry previouslyassigned in a 2-D projection (4). Symmetry was imposed on thevolume after calculation of the center of gravity and the correctaxis of symmetry. Volumes were subsequently displayed by usingeither WEB (14) or XFIT (29).

Resolution determination. For a particle in the size range of PilQ, $~$ 300 particles wouldbe required to provide a complete data set to 2.5-nm resolution(12): the data set of 503 particles with symmetry averagingapplied exceeds this practical minimum requirement. The resolutionof the final volume was calculated by determining the resolutionbetween two subvolumes by using either Fourier ring correlation(FRC) criteria (43) with a cutoff point of FSC (Fourier shellcorrelation) = 0.5 (to give a value of 2.1 nm) or by employingthe differential-phase residual (DPR) method (35) (to give avalue of 2.8 nm). The mode by which resolutions are reportedfor 3-D volumes is currently under debate because differentmethods provide slightly different resolution values (11). Resolutioncalculations made by the FRC method tend to provide more optimisticresolution values than those obtained by the DPR method. Toaccount for this fact, we report a mid-range resolution valueof 2.5 nm from the two methods currently recommended as bestpractice (12). An alternative method to convert FRC resolutionvalues to their corresponding SNR (signal-to-noise ratio) valueshas been proposed, although the use of an FSC of 0.5 as a cutoffpoint has been supported by comparisons of cryo-EM and X-raystructures of adenovirus type 2.

Tfp modeling. A 3-D homology model for the N. meningitidis pilin subunit wasbuilt based on the X-ray crystal structure of the homologousN. gonorrhoeae pilin subunit (PDB accession code 2PIL), by usingthe Modeller program (41). The amino acid sequences of the twoproteins are 86% identical and 89% similar. The N. meningitidispilus fiber was then built by applying the transformations givenin the 2PIL PDB header, based on the pilus fiber model withfivefold helical symmetry described previously (36).

RESULTS

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Results

PilQ complex purification. The yield of native oligomeric PilQ complex purified from meningococcalmembranes was significantly improved by using the capsule negativeN. meningitidis M1080 mutant also expressing truncated lipooligosaccharideas the protein source. Improvements in the purification procedureof the PilQ complex were also achieved by using the detergentZwittergent 3-10 (rather than SDS) to solubilize outer membranefractions. This procedure produced PilQ samples equivalent inpurity to those of previous materials (4) but at the higherprotein concentration required for extensive EM analyses.

EM and SPA of PilQ. A representative electron micrograph tilt pair of negativelystained PilQ complexes that were used to calculate the final3-D volume is shown in Fig. 1A. The Zwittergent-solubilizedPilQ complexes, indicated by arrows, presented themselves primarilyas circular donut-like rings of protein density that encloseda central stain-excluding cavity. We also observed a distinctiveminority population of particles corresponding to a varietyof side-on and partial side-on views of PilQ (Fig. 1B). Theseviews were rare, however, and were not present in sufficientnumbers for either accurate 2-D projection mapping or the calculationof a 3-D structure. It is possible that these particles correspondto pairs of PilQ dodecamers; similar behavior has been observedin at least one other secretin (34). SPA of the donut-like projectionsresulted in the identification of two highly related principalgroups from the data set (Fig. 1C). Subsequent analysis of thesegroups revealed that they only differed slightly in their Eulerangle orientations (Table 1), and these were applied duringvolume merging. The class-averaged projection maps for thesegroups demonstrated that the PilQ complexes were identical,with regard to the overall complex features and distributionof negative stain, to SDS-solubilized PilQ preparations observedpreviously (4). Examination of the selected PilQ particles revealedthat the central cavity measured 6 to 6.5 nm across, with anouter diameter of 15.5 nm, which was at the lower end of theprevious size distribution (15.5 to 16.5 nm) (4). Since theinner ring diameter was identical to previous distributions,the smaller outer diameter changes were likely to be causedby a different detergent annulus around the circumference ofthe complex. As with previous PilQ preparations, we found thatthe protein adsorbed to EM grids with a low affinity, requiringprotein concentrations greater than 100 µg/ml and incubationtimes exceeding 10 min (4). This effect may be due to the highestimated pI of the protein (9.9), which could lead to a poorcharge interaction at the grid surface (50).

Oligomeric form of PilQ. Our prior work on the structure of PilQ demonstrated that 2-Dprojection maps of the PilQ complex possessed 12-fold rotationalsymmetry (4). This symmetry was also observed in the volumecalculated from merging the two main groups shown in Fig. 1C:the 12-fold symmetry was readily apparent from the accumulationof 12 negative stain density peaks (numbered 1 to 12) outsidethe periphery of the ring of the calculated 3-D volume (Fig.2). These peaks occurred at approximately 30° intervals,although some variation from the "ideal" positions was apparentbecause no symmetry had been applied to the volume. In agreementwith these data, self-orientation and power spectrum analyseswere performed on the 3-D reconstruction, and the findings wereconsistent with data previously reported (not shown). It isinteresting that the accumulation of these stain peaks is primarilylocated on the outside of the PilQ ring in the 3-D volume. Since,in this orientation, the complex is positioned on the grid withthe ring facing down, this observation suggests that the upperand side portions of the ring are more accessible to negativestain than the constricted section of the complex. Impositionof any symmetry other than 2-, 3-, 4-, 6-, or 12-fold smoothedout the particle features observed and/or skewed the surfacerelief of the central cavity, a finding consistent with theassignment of 12-fold symmetry.

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FIG. 2. The distribution of accumulated negative stain around the circumference of the merged PilQ volume demonstrates 12-fold symmetry. The unsymmetrized PilQ volume (n = 503) displayed as a density map (in blue at 2 ${sigma}$ above the mean density value) is positioned to correspond to the donut-like projections in Fig. 1. Twelve peaks of negative-stain volume (shown in red and yellow at 1.5 ${sigma}$ and 2 ${sigma}$ , respectively, above the mean negative-stain density) occur at approximate 30° intervals around the PilQ ring and are labeled 1 to 12. The boundary between the regions in the volume corresponding to the PilQ complex and the surrounding stain pockets is shown by the dotted line. Scale bar, 10 nm.

3-D structure of PilQ. Figure 3A shows the surface relief of the final PilQ volumewith 12-fold rotational symmetry applied and presented at 2 ${sigma}$ above the mean density. Symmetry averaging is a powerful techniqueand has been used to successfully analyze the structures ofmany virus and helical assemblies (10, 14). This view correspondsto the face-on or donut 2-D projection of PilQ (Fig. 1C) andclearly shows the 15.5-nm ring of the 12 subunits enclosinga deep central cavity 6.2 to 6.5 nm in diameter. Inspectionof the cavity from this view reveals that the impression isnot continuous through the height of the complex and significantdensity could be identified in the bottom. Around the circumferenceof the ring, 12 repeating surface protrusions were apparentat $~$ 3-nm intervals, and each of the 12 monomers were estimatedto contribute an area of ca. 4.5 by 3.0 nm to the upper surfaceof the PilQ ring. Figure 3B shows the surface relief of PilQafter a rotation of the face-on view of PilQ by 90° aroundthe y axis. This side view reveals that the PilQ complex possessesa distinctive two-region structure, with an overall height of $~$ 12 nm and a rounded conical appearance. The upper portion ofthe structure forms a discrete ring (R region) with a diameterof 15.5 nm and a height of 8 nm. The regular detail observedaround the top of the ring also continues around the side at3-nm intervals. Below this ring is a smaller conical plug (theP region) that gradually tapers toward a flattened point. Theinterface between the R and P regions sharply constricts from15.5 to 11 nm and then gradually tapers to a blunt point 4.5nm in diameter over the region height of 4 nm. Figure 3C reproducesthe PilQ volume shown in Fig. 3B but with 50% of the foremostvolume removed to reveal the interior relief of the complex.The cavity has a distinctive funnel-like shape that is noncontinuous,with an overall height of ca. 10 nm. The majority of the cavityis located in the R region and gradually closes to a fixed point $~$ 3 nm in diameter ca. 2 nm into the P region. To provide a senseof scale for the Tfp substrate, a homology model of the N. meningitidispilus fiber (shown in two views related by a 90° rotation)is included at the same scale as PilQ. Given that only one turn(five subunits) of the pilus fiber is shown, it is clear thatthe dimensions of the cavity are too small to accommodate afully assembled pilus fiber running continuously through thePilQ complex in the purified conformation.

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FIG. 3. Surface-rendered 3-D volumes of PilQ with 12-fold rotational symmetry applied. Volumes are displayed at a threshold of 2 ${sigma}$ above the mean volume density. Scale bar, 10 nm. (A) Face-on view corresponding to the donut-like projection in Fig. 1. (B) Rotation of the face-on view of the PilQ complex 90° through the y axis. Viewed from the side, the PilQ volume has two distinct regions: a ring (R) region connected to an underlying plug (P) region. (C) A cross-sectional side-on view of the PilQ volume with 50% of foremost volume removed through the z axis to reveal the structure of the internal cavity. The constriction of the central cavity may be seen at the bottom of the complex and is indicated by arrows. Ribbon plots of the atomic model of the N. meningitidis Tfp fiber are displayed to the right in a side-on and face-on view at the same scale as the PilQ volumes. Five subunits (one helical turn) are shown.

It should be noted that structural information calculated byusing random conical tilt methodology applied to negativelystained particles has two main limitations. First, a negativestain, although providing excellent contrast, a high particlesignal/noise ratio, electron beam stability, and experimentalreproducibility, also has several well-documented disadvantagesthat may affect a sample to greater or lesser degrees (19).These problems include uneven stain penetration of the particle,sample deformation by shrinkage, flattening and skewing, anda practical resolution limit of $~$ 2 nm. Despite these constraints,negative staining remains a useful technique for acquiring moderateresolution structural information of a biological complex, andcomparisons of several structures obtained by using negativestaining versus unstained cryo data do demonstrate good overallstructural correlation (12). The second issue relates to themaximum practical tilt angle used ( $~$ 60°) when a specimenis tilted in a transmission electron microscope. This limitationresults in the loss of some information along the z axis or"a missing cone of data" in the back-projected volume (12).For any 3-D reconstruction calculated in this way, this phenomenonresults in a lower resolution for structural features extendingin the z direction, perpendicular to the specimen support film.For the PilQ structure presented here this means that the resolutionof detail through the height of the complex is reduced slightly.We report an overall resolution of 2.5 nm for the PilQ structurebut it is important to note that, even if we consider the lowerDPR resolution estimate of 2.8 nm, this resolution remains morethan sufficient to resolve all of the structural features ofthe PilQ complex discussed here.

DISCUSSION

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Discussion

The determination of the 3-D volume of PilQ has a number ofimportant implications for secretion processes in general andthe mechanism of Tfp biogenesis in particular. Previous workhas established that purified secretin complexes form gatedconductance channels when reconstituted into lipid membranes(3, 27, 31). The structure presented here would establish astructural explanation for this phenomenon, providing evidencefor a "plug" or constriction in the channel that is predictedto run across the outer membrane. It is clearly advantageousfor the bacterial cell to regulate the accessibility of pilinsubunits to PilQ since this secretin is expressed constitutivelyat high levels (50) and a persistently open 6.5-nm channel wouldallow periplasmic proteins and other valuable periplasmic moleculesto diffuse away from the cell. A limitation of the current structureis that it does not assign the outer or inner faces of the complex.We presume that the open mouth of the cavity faces outward fromthe cell because it needs to house the Tfp as it is extruded,but there is no direct evidence for this supposition in thedata presented here. If this is indeed the case, it would suggestthat the constricted end of the channel lies on the periplasmicside of the outer membrane. This is an attractive propositionbecause it would allow this region of PilQ to interact withother Tfp biogenesis proteins from the periplasm and inner membrane.

It is also tempting to speculate on how PilQ might interactwith the growing or retracting Tfp fiber. The structure presentedhere suggests two possibilities. The first is that assemblyof the pilus fiber occurs outside the PilQ cavity and that thefiber is extruded through the protein in its intact form. Inorder for this to happen, the dimensions of the cavity wouldneed to expand marginally, possibly through a conformationalchange induced by interactions of PilQ with other protein components,and the P region would need to "unfurl." The second possibilityis that the site of pilus formation is actually inside the PilQcavity itself and that individual pilin subunits are deliveredor removed from this site depending on the relative activitiesof Tfp assembly or retraction proteins such as PilT. It is notpossible, on the basis of these structural data, to distinguishbetween these alternate models, although it is interesting thatthey both require a substantial degree of conformational flexibilityof the P region of the molecule. It is important to emphasize,as a caveat, that the results presented here were obtained fromdetergent-solubilized, purified protein and that extractionof PilQ from the meningococcal membrane and removal of otherprotein components could affect the conformation or structureof the protein. Another important question is the position ofthe outer membrane: again, this is impossible to discern fromthese observations on detergent-solubilized material, althoughthe size and morphology of the R region suggest that this portioncould contain the membrane-spanning portions of the complex.

Our structure of PilQ can be compared to the low-resolutionstructures of two other secretin complexes. Scanning transmissionEM observations of two orientations of the pIV filamentous phagechannel showed that the complex formed a cylindrical assemblyof 12 to 14 monomers (26). More recently, the structure of thepIV multimer has been determined to a 22-Å resolutionby cryo-EM (34). The pIV complex exhibits 14-fold, rather than12-fold, rotational symmetry and forms a barrel-like structure.The channel through the molecule is blocked, as is the casewith PilQ, but in a central portion of the pore, rather thanat one end. Another secretin that has been studied in some detailis the PulD-PulS complex from K. oxytoca (31). The results showedan asymmetric cylindrical complex composed of 12 monomers witha height of 19 nm and a diameter of 25.8 nm. Around the peripheryof the complex 12 separate radial densities were observed: thesefeatures were proposed to originate from the secretin assemblylipoprotein PulS. The central cavity was large in diameter ( $~$ 7.5to 8.0 nm) and apparently continuous throughout the complex,although there was biochemical evidence that the channel wasgated (31). Further observations of PulD-PulS in negative stainhave shown a side-on view of the complex, in a dimeric organizationwith pseudo-mirror symmetry (32). Given that PulD and PilQ exhibithomology only within the C-terminal domains of their genes,it is possible that some of these structural differences areattributable to variations in the structure of the N-terminalhalf of each protein.

By providing novel information on the pore cavity, the PilQstructure presented here represents a first step toward an understandingof the structure of the Tfp biosynthetic apparatus. There aremany intriguing aspects of this complex molecular machine, notleast of which are questions regarding how pilus extrusion isregulated and how the extension process is reversed to retraction.Clearly, 3-D studies provide a significant contribution towardexplaining these phenomena.

ACKNOWLEDGMENTS

This work was supported by grants from the Wellcome Trust andthe Norwegian Research Council.

We thank Stephan A. Frye for advice in the PilQ complex purificationprocess, Matthias Frosch for providing the plasmid pMF121, G.Verlarde for technical assistance with image processing, andK. Sidhu for software support.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biomolecular Sciences, UMIST, P.O. Box 88, Manchester M60 1QD, United Kingdom. Phone: 44-0161-200-4207. Fax: 44-0161-236-0409. E-mail: Jeremy.Derrick{at}umist.ac.uk.

REFERENCES

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