Bacterial pili are involved in a host of activities, including motility,adhesion, transformation, and immune escape. Structural studies of thesepili have shown that several distinctly different classes exist, with nocommon origin. Remarkably, it is now known that the archaeal flagellarfilament appears to have a common origin with the bacterial type IV pilus,and assembly in both systems involves hydrophobic N-terminal α-helicesthat form three-stranded coils in the center of these filaments. Recent workhas identified further genes in archaea as being similar to bacterial type IVpilins, but the function or structures formed by such gene products wasunknown. Using electron cryo-microscopy, we show that an archaeal pilusfromMethanococcus maripaludis has a structure entirely different from that ofany of the known bacterial pili. Two subunit packing arrangements wereidentified: one has rings of four subunits spaced by ∼44 Å and the other hasa one-start helical symmetry with ∼2.6 subunits per turn of a ∼30 Å pitchhelix. Remarkably, these schemes appear to coexist within the samefilaments. For the segments composed of rings, the twist between adjacentrings is quite variable, while for the segments having a one-start helix thereis a large variability in both the axial rise and the twist per subunit. Since thispilus appears to be assembled from a type IV pilin-like protein with ahydrophobic N-terminal helix, it provides yet another example of howdifferent quaternary structures can be formed from similar building blocks.This result has many implications for understanding the evolutionarydivergence of bacteria and archaea.
Keywords: helical polymers; polymorphisms; cryo-EM; quaternary structure;scanning transmission EM
Introduction
Archaea possess various kinds of cell surfaceorganelles that differ substantially from those ofbacteria.1 Archaeal flagella have been characterizedonly recently,2–7 and all evidence supports the notionthat the archaeal flagellar filament is related to thebacterial type IV pilus.8–10 However, no structural orgenetic information is available on archaeal pili,which are observed on the surfaces of many archaealspecies.11–13 The structure and cellular functions ofarchaeal pili are unknown, as is the possibility of
ess:
ing transmissione helical real spacer function.
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whether there are different types of pili found inArchaea, as is well known for bacteria. Recently, agenomic analysis identified a diverse group ofarchaeal proteins that contain class III signalpeptides, similar to those found in bacterial type IVpilins and archaeal flagellins, and suggested thatthese might assemble into cell surface structures,such as pili.14
Results
Helical symmetry estimates
We have examined pili from the archaeon Metha-nococcus maripaludis by both negative stain and cryo-EM of frozen-hydrated samples. The pili wereisolated following detergent extraction of cells andprecipitation by polyethylene glycol. The filament
appears by both negative stain (Fig. 1a) and cryo-EM(Fig. 1b) as a thin and featureless rod. Filamentsappeared thinner by negative stain than they did inthe frozen-hydrated state. Since we show (below)using cryo-EM that there is a significant hollowlumen in these filaments, the lack of significant stainpenetration into the central lumen in images ofnegatively stained filaments is probably due to theflattening of these filaments, so that the lumen iscrushed.Despite the radial shrinkage and likely flattening
in negative stain, the negatively stained filamentswere useful in estimating the symmetry of the pili.We extracted 1907 non-overlapping segments (each300 pixels or 1260 Å in length) from images of thenegatively stained filaments (Fig. 1a), and thenadded together the power spectrum from eachsegment (Fig. 1c). This method is independent ofany alignment needed to average together images,and is thus unbiased.15 The resulting power spec-trum shows three layer-lines. Due to the distance ofthe peaks from the meridian, and the diameter of thefilaments, the layer-line at ∼1/(17 Å) (cyan line)
Fig. 1. Electron micrographs and helical symmetry estimfrozen-hydrated archaeal pili (b). The scale bar (b) is 500 Å. (coverlapping segments (each 300 pixels or 1260 Å in length)arrows): 1/(17 Å), 1/(44 Å) and 1/(58 Å). The logarithm of theallow all three layer-lines to be visible simultaneously. (d) M1618.4±8.8 (SEM) Da/Å.
must arise from a one-start helix. The layer-line at∼1/(44 Å) (upper red line) is a meridional layer-linecorresponding to the reciprocal of the axial rise perasymmetric unit. There is intensity on this layer-lineon the meridian, but the strongest peaks are off themeridian and must correspond to the secondmaximum of a Bessel function of order 0. Thelayer-line at ∼1/(58 Å) (lower red line) arises fromeither a three-start helix or a four-start helix, basedupon the distance of the peak from themeridian. Theapparent molecular mass, determined by SDS-PAGE, of the major pilus subunit is ∼17 kDa (S.Y.M.N. et al., unpublished results). Given the relativelysmall size of the subunit, it is inconceivable that onecould have ∼0.4 subunit per turn of the 17 Å helix(resulting from the observed 44 Å axial rise perasymmetric unit). On the other hand, if there is apoint group symmetry (so that the axial rise perasymmetric unit corresponds to a ring of subunits),onewould not observe a layer-line at 1/(17Å) arisingfrom a one-start helix. The most likely explanation ofthis averaged power spectrum is therefore that thepili are amixture of point group and one-start helical
ates. Electron micrograph of negatively stained (a) and) An averaged power spectrum generated from 1907 non-of negatively stained pili shows three layer-lines (blackintensity has been taken to reduce the dynamic range andass per unit length measurements yielded an average of
Fig. 2. Sorting based on C4 symmetry and one-starthelical symmetry. The 28,577 segments from the cryo-EMimages were sorted into two groups by models with eitherC4 symmetry or one-start helical symmetry; 54% of thesegments were sorted to have point group symmetry,while the remaining 46% were sorted to have a one-starthelix. The averaged power spectrum for the segmentssorted as having C4 symmetry (a) has a meridional layer-line (red arrow) at 1/(44 Å). The averaged powerspectrum for the segments sorted to have one-start helicalsymmetry (b) is much poorer, which suggests this group ismore heterogeneous than the other group. The broad yetweak layer-line at 1/(56 Å) (red arrow) can be interpretedas either n=2 or n=3, based upon the distance of the firstpeak from the meridian and the diameter of the filaments.
458 The Structure of an Archaeal Pilus
symmetries, and this averaged power spectrum isshowing a superposition of these two symmetries.In order to establish the helical symmetry for
each state, mass per unit length was measuredusing scanning transmission electron microscopy(STEM).16 The STEM results yield a mass per unitlength of 1618.4±8.8 (SEM) Da/Å (Fig. 1d). Thisshows that the averaged axial rise per subunit is∼10.5 Å. The distribution is not obviously bimodal,showing that the two different helical symmetriespresent must have comparable mass per unitlength values. In the case of the one-start helicalsymmetry (with ∼17 Å as suggested by the powerspectrum of negatively stained filaments), theSTEM measurements suggest ∼1.6 subunits perturn. In the case of the point group symmetry, theaxial rise per asymmetric unit would be ∼32 Å forC3 symmetry or ∼42 Å for C4 symmetry. Since theaxial rise per asymmetric unit as suggested by theaveraged power spectrum of negatively-stained fil-aments is ∼44 Å (Fig. 1c), the point group symmetrymust be C4.
Sorting the filaments and 3D reconstruction
We used the iterative helical real space reconstruc-tion (IHRSR) method to reconstruct the pili fromcryo-EM images.17,18 The IHRSR approach has beenshown to be a powerful method for reconstructingfilaments that are flexible,19 heterogeneous,20 sufferfrom Bessel overlap,21 or are weakly diffracting.22
An interesting question is what happens if oneignores the structural heterogeneity and attempts touse IHRSR on the heterogeneous population offilament segments. We started the procedure with28,577 overlapping segments (each 480 Å in length)extracted from cryo-EM images of the pili. TheIHRSR approach failed to converge to the samesolution from different starting points using either ofthe two symmetries described. This is consistentwith our finding that the population must beheterogeneous, and that the failure to achieveconvergence in IHRSR is due to heterogeneity.15,22
An iterative approach was then used to sort thesegments into two groups: C4 symmetry segmentsand one-start helical symmetry segments. First, afeatureless stacked disk structure with spacings of44 Å between disks and a continuous one-start helixwith a pitch of 17 Å were used as references for aninitial sorting. Two groups were generated on thebasis of different subunit packing schemes. Recon-structions were generated independently from thetwo groups of segments. These two reconstructionswere then used as references for a new sorting. Withthese new references, 54% of the segments werecharacterized as having C4 symmetry, while theremaining 46% were characterized as having one-start helical symmetry. The validity of the sortingwas confirmed by the differences between theaveraged power spectra from each group (Fig. 2aand b). The averaged power spectrum for the groupsorted as having point group symmetry (Fig. 2a) hasa meridional layer-line (red arrow) at 1/(44 Å). This
layer-line is strong and thin, suggesting the axial riseis relatively fixed in this group. The n=4 layer-line isnot visible, suggesting that the twist between adjacentrings is variable. The averaged power spectrum forthe group sorted as having one-start helical symme-try (Fig. 2b) has a layer-line at∼1/(56 Å) that is weakand broad. The n=1 layer-line, observed in negativestain at ∼1/(17 Å), is not visible in the cryo-EMimages, which have a much lower signal-to-noiseratio than those in negative stain. We can show (seebelow) that the failure to see a layer-line at∼1/(17 Å)is because of additional heterogeneity in symmetrywithin this group. Further heterogeneity was sug-gested by the fact that the IHRSR approach failed toconverge to the same solution from different startingpoints in each group (C4 and one-start symmetries).We therefore attempted to sort each of the two groupsinto more homogeneous subgroups.
Fourfold rotational symmetry
As suggested by the averaged power spectrum ofthe first group (Fig. 2a), we expected that mostvariation is in the twist between adjacent rings. We
459The Structure of an Archaeal Pilus
therefore attempted to use variants of a globalreconstruction with different twist imposed asreferences to sort this group. The variability intwist observed was quite large, with a range in theorder of 56°–88° twist per subunit (data not shown).Although segments can be sorted into subgroupsthat differ in twist, that does not necessarily meanthat the sorting is valid. Several tests can be done toshow that the sorting accurately reflects an intrinsicvariability in the data. If we take the segmentsclassified as having a twist closest to 64°, and startthe IHRSR algorithm with an initial twist near thepopulation mean, 72°, does the symmetry convergeback to a twist of ∼64°? Similarly, if we take thesegments classified as having a twist closest to 80°,and again start the iterations with a twist of 72°, do
Fig. 3 (legend on next page)
the cycles converge to a value near 80°? This wasindeed the case, arguing that the sorting based uponvariability in twist was valid.A second, and independent, test for the proposed
variability in twist comes from the power spectra. Ifthere is no change in axial rise from ∼44 Å, and thevariability is almost entirely in the twist betweenrings, then one can predict the change in layer-linespacings that should be seen as the twist changes.Since the structure has C4 symmetry, the pitch of thefour-start helices should be (Δz×360°)/Δφ, whereΔz is the axial rise per subunit andΔφ is the twist persubunit along the four-start helix. The position of then=4 layer-line should therefore be (4 ×Δφ)/(Δz×360). For the subgroup with Δφ=64°, theexpected n=4 layer-line should be at 1/(61 Å);
460 The Structure of an Archaeal Pilus
when Δφ=72°, the n=4 layer-line should be at1/(55 Å); and when Δφ=80°, the n=4 layer-lineshould be at 1/(49 Å). Such a large shift should bereadily visible. Generating separate power spectrafrom the segments initially classified as having a twistclosest to either 64°, 72° or 80° (Fig. 3a–c, left halves),the layer-lines shift exactly as predicted. Since there isno change in the distance of the peaks from themeridian of the transform as the layer-line spacingschange,we can eliminate the trivial possibility that thechanges we are seeing are simply due to changes inmagnification. This variation explains why the n=4layer-line is not visible in the global power spectrumof this group (Fig. 2a), and why smaller subsets aftersorting generate better power spectra than can bedone globally.We used the IHRSR method to reconstruct the
three most populated subgroups. The reconstructionfrom the segments classified as having a twist of∼64° (containing 14% of this group) (Fig. 3d) has C4symmetry with an axial rise of 43.9 Å and twist of65.0° (or −25°) per subunit between adjacent rings.The reconstruction from the segments classified ashaving a twist of∼72° (containing 21% of this group)(Fig. 3e) has C4 symmetry with an axial rise of 43.8 Åand a twist of 72.0° (or −18°) per subunit betweenadjacent rings. The reconstruction from the segmentsclassified as having a twist of ∼80° (containing 12%of this group) (Fig. 3f) has C4 symmetry with an axialrise of 43.5 Å and a twist of 80.1° (or −9.9°) persubunit between adjacent rings. We do not know theabsolute hand of these reconstructions, so the 65° (or−25°) set might actually be −65° (or +25°). We willuse the convention that the hand is as shown, untilsuch time as the absolute hand can be determined,for example by having an atomic structure of thesubunit or by surface shadowing. The power spectragenerated from the projections of each reconstruc-tion (Fig. 3a–c, right halves) match the power spectraof the corresponding subgroups (Fig. 3a–c, lefthalves) very well, which supports the validity ofthe reconstructions and the sorting. While the
Fig. 3. Sorting of the group with C4 symmetry. The 15,297by differences in the angular rotation between adjacent ringsmost populated subgroups (a–c, left half) are much improved(Fig. 2a), suggesting reduced heterogeneity in each subgroup.−4 layer-lines shift as expected in the three subgroups (a–c, re∼64.0° and an axial rise of ∼43.8 Å. The averaged power splayer-line at 1/(44 Å), n=+4 layer-line at 1/(60 Å) and n=−subgroup has C4 symmetry with an axial rise of 43.9 Å and atwist of ∼72.0° and an axial rise of ∼43.8 Å. The averaged pon=0 layer-line at 1/(44 Å), n=+4 layer-line at 1/(53 Å) and nhas C4 symmetry with an axial rise of 43.8 Å and a twist of∼80.0° and an axial rise of ∼43.8 Å. The averaged power spectline at 1/(44 Å), n=+4 layer-line at 1/(48 Å) and n=+3 at 1symmetry with an axial rise of 43.5 Å and a twist of 80.1°. The(a–c, right half) match the corresponding averaged powerreconstructions from the three subgroups are very similar, excbeen chosen to enclose 100% of the expectedmolecular volume.asymmetric subunits is along a left-handed four-start helix (e,which one subunit in each has been aligned (black arrow) show(red arrow). The yellow reconstruction has a twist of 65.0° and thcontour plots that are spaced 24 Å away from each other have
different reconstructions differ in the relative twistbetween adjacent rings, they all show a very similarsubunit, similar “fenestrations”, and a very similarmodulation of the central lumen. In all three recon-structions the connectivity between asymmetricsubunits is along a left-handed four-start helix (Fig.3e, red line).The lumen at some axial levels is quiteconstricted (Fig. 3h), while it is quite broad at otheraxial levels (Fig. 3i). The outer diameter of all thereconstructions is ∼85 Å, so the large variability intwist does not introduce any variability in diameter.The variable twist also does not introduce anyvariability in the mass per unit length, which isexpected to be ∼1.56 kDa/Å in this state. This valueis quite consistent with the STEM data (Fig. 1d).Using the 0.5 Fourier shell correlation criterion, we
find that the resolution of the reconstructions is∼20 Å. This estimate is conservative because itcompares two independent data sets, in contrast tothe standard Fourier shell correlation method thattypically compares two halves of a single data setprocessed by identical methods and aligned to thesame reference.23 We think that the main limitationon resolution is the continuous variability of thetwist, so that while each of the three sets is morehomogeneous than the original population, they arestill rather heterogeneous.
One-start helical symmetry
What happens in the group that was sorted to haveone-start helical symmetry when we try to furtherdecompose variability? The poor power spectrum ofthis group (Fig. 2b) suggests strong heterogeneity inthe symmetry.We therefore attempted to use variantsof a global reconstruction with different symmetries(angular rotation and axial rise per subunit) asreferences. We used nine arbitrarily chosen symme-tries, with three different angles (211°, 216°, and 221°)and three different axial rises (10.7, 11.7, and 12.7Å).Amulti-reference sorting against projections of thesereference volumes was used to classify image
segments of the first class were sorted into five subgroupsof subunits. The averaged power spectra from the threecompared to the averaged power spectrum before sortingThe meridional layer-line is fixed while the n=+4 and n=d lines). The first subgroup was sorted to have a twist ofectrum (a, left half) can be interpreted as having an n=04 layer-line at 1/(144 Å). (d) The reconstruction of thistwist of 65.0°. The second subgroup was sorted to have awer spectrum (b, left half) can be interpreted as having an=−4 at 1/(204 Å). (e) The reconstruction of this subgroup72.0°. The third subgroup was sorted to have a twist ofrum (c, left half) can be interpreted as having an n=0 layer-/(350 Å). (g) The reconstruction of this subgroup has C4power spectra from the projections of each reconstructionspectrum of each subgroup (a–c, left half). (d–g) The
ept for the difference in twist. The surface thresholds haveIn all three reconstructions, themain connectivity betweenred line). (g) A superposition of the two reconstructions ins a 15.1° difference in subunit twist at a subunit∼44Å awaye cyan one has a twist of 80.1°. (h and i) Two cross-sectionaldifferent outer diameters and different size lumens.
461The Structure of an Archaeal Pilus
segments into nine different subgroups. Separatepower spectra of segments sorted into three repre-sentative subgroups (Fig. 4a–c, left halves) showsignificant shifts of the layer-lines. The layer-linescorresponding to one-start helices are not visible,probably because of further variability of pitchwithinthe subgroups and the form of the averaged contrasttransfer function (CTF) (see below).We used the IHRSR method to reconstruct three
subgroups, classified ashavinga twist of∼221.0°, thatgenerated reasonable power spectra. The reconstruc-tion (Fig. 4d) from the segments classified as havingan axial rise of ∼10.7 Å and a twist of ∼221.0° (con-taining 11% of this group) converges to a one-starthelical symmetry with an axial rise of 10.8 Å and a
Fig. 4 (legend o
twist of 220.9° per subunit. This twist is defined alonga right-handed one-start helix thatwould have a pitchof 17.6 Å (10.8 Å×360°/220.9°). There would also beleft-handed one-start helix in this symmetry that has apitch of 28.0 Å (10.8 Å×360°/139.1°). The reconstruc-tion (Fig. 4e) from the segments classified as having anaxial rise of∼11.7Å and a twist of∼221.0° (containing11% of this group) converges to a one-start helicalsymmetry with an axial rise of 11.7 Å and a twist of221.1° per subunit (yielding a right-handed one-starthelixwith a pitch of 19.1 Å and a left-handed one-starthelix with a pitch of 30.1 Å). The reconstruction (Fig.4f) from the segments classified as having an axial riseof ∼12.7 Å and a twist of ∼221.0° (containing 12% ofthis group) converges to a one-start helical symmetry
n next page)
462 The Structure of an Archaeal Pilus
with an axial rise of 13.0 Å and a twist of 221.1° persubunit (yielding a right-handed one-start helix witha pitch of 21.2 Å and a left-handed one-start helixwith a pitch of 33.7 Å). Each of these three valuesshown for the axial rise (10.8, 11.7 and 13.0 Å) isconsistent with the mass per unit length histogram(Fig. 1d).The agreement between the sorting and the
IHRSR reconstructions supports the validity of thesorting, as do the good matches between the powerspectra of projections of each reconstruction (Fig.4a–c, right halves) and the power spectrum of eachsubgroup (Fig. 4a–c, left halves). Cross-sectionalcontour plots of the second reconstruction (Fig. 4hand i) show a rather constant lumen, in contrast tothe large modulation of the lumen with C4 symme-try. The outer diameter is ∼85 Å. The surface viewsof the three reconstructions (Fig. 4d–f) are similar,except for the differences in axial rise (Fig. 4g). Ineach of the three reconstructions, the connectivitybetween subunits is along a right-handed two-starthelix (Fig. 4e, cyan line) and a left-handed three-starthelix (Fig. 4e, red line). Using the 0.5 FSC criterion,we find that the resolution of the reconstructions is∼20 Å. We think that the main limitation onresolution is the continuous variability of the axialrise, in contrast to the C4 symmetry segments wherethe main variability is in the twist. This variabilityalso explains why the layer-line arising from the left-handed one-start helix is not visible at ∼1/(18 Å)−1/(21 Å) in the power spectra from the cryo-EMimages (Fig. 4a–c), although a layer-line is seen at∼1/(17 Å) in the global power spectrum generatedfrom negatively stained filaments that have a muchhigher signal-to-noise ratio. The change in spacingfrom the observed 1/(17 Å) in negative stain to thepredicted 1/(18 Å) − (1/(21 Å) in ice is consistentwith the shrinkage that we see radially in negativestain when compared to cryo-EM. Similarly, we donot see a layer-line arising from the right-handedone-start helix at ∼1/(28 Å) −1/(34 Å) due to both
Fig. 4. Sorting of the group with one-start helical symmetrynine subgroups by differences in the angular rotation and axialthree different angles (211°, 216°, and 221°) and three different apower spectra of three representative subgroups (a–c, left hapower spectrum before sorting (Fig. 2b), suggesting reduceddifferent subgroups (a–c, red lines) as expected. The first subgrof∼10.7 Å. The averaged power spectrum (a, left half) can be in+3 at 1/(66 Å). The reconstruction of this subgroup (d) hassubgroup was sorted to have a twist of ∼221.0° and an axial rcan be interpreted as having an n=−2 layer-line at 1/(50 Å) anhas an axial rise of 11.7 Å and a twist of 221.1°. The third subgrof∼12.7 Å. The averaged power spectrum (c, left half) can be in+3 at 1/(82 Å). (g) The reconstruction of this subgroup has an afrom projections of each reconstruction (a–c, right half) matchThe surface threshold for each of the reconstructions has been(d–g) The reconstructions from the three subgroups are veryreconstructions, the connectivity between subunits is alonghanded three-start helix (e, red line). (g) A superposition of twhas been aligned (black arrow) shows a 13.2 Å difference in areconstruction has an axial rise of 10.8 Å and the cyan onecontour plots spaced 9.6 Å away from each other show simi
variability as well as the fact that this would be at aspacing that is near a minimum in the averaged CTF(Fig. 4d–f).
Comparison of the two symmetries
A superposition of the reconstructions with C4symmetry and one-start helical symmetry (Fig. 5a)in which one subunit in each has been aligned (blackarrow) shows the similarity of the structure in termsof the appearance of single subunits, but hugedifferences in helical symmetry. The difference inhelical symmetry can be understood by looking atthe helical nets (Fig. 5b and c), which are diagramsshowing the helical lattice on the surface of acylinder. For the filaments with C4 symmetry, themain connectivity between subunits is along a left-handed four-start helix. For the filaments with one-start helical symmetry, the connectivity switches tothe left-handed three-start helices and right-handedtwo-start helices. The switch in connectivity may bea way of creating an elastic filament out of basicelements that are themselves relatively inextensible.Several obvious questions are raised by our
results. One is whether individual pili are in onesymmetry state (either C4 or one-start helix) and theswitching in symmetry is between filaments, andnot within filaments. We have tried to answer this bylooking at the four longest pili in our data, andclassifying segments from these filaments usingmultiple references. Each of these filaments sug-gested that both states are present within individualfilaments. Another question is whether the varia-bility in both helical symmetries is due to two orthree discrete states, or arises from a continuum(noting that we would be unable to distinguishbetween many discrete states and a continuum). Bylooking at different bins generated by sortingagainst multiple references, all indications are thatthe variability represents a continuum. This isreflected by the fact that the layer-lines in the
. The 13,280 segments of the second group were sorted intorise per subunit. Nine arbitrarily chosen symmetries, withxial rises (10.7, 11.7, and 12.7 A°) were used. The averagedlf) are much improved compared to the averaged globalheterogeneity in each subgroup. The layer-lines shift inoup was sorted to have a twist of ∼221.0° and an axial riseterpreted as having an n=−2 layer-line at 1/(47 Å) and n=an axial rise of 10.8 Å and a twist of 220.9°. The secondise of ∼11.7 Å. The averaged power spectrum (b, left half)d n=+3 at 1/(72 Å). e, The reconstruction of this subgroupoup was sorted to have a twist of ∼221.0° and an axial riseterpreted as having an n=−2 layer-line at 1/(56 Å) and n=xial rise of 13.0 Å and a twist of 221.1°. The power spectrathe averaged power spectrum of each class (a–c, left half).chosen to enclose 100% of the expected molecular mass.similar, except the difference in axial rise. In all the threea right-handed two-start helix (e, cyan line) and a left-o reconstructions (d and f) in which one subunit in each
xial rise at a subunit ∼70 Å away (red arrow). The yellowhas an axial rise of 13.0 Å. (h and i) Two cross-sectionallar outer diameters and lumen size.
Fig. 5. Comparison between the two subunit packing schemes. (a) A superposition of the reconstructions with C4symmetry and one-start helical symmetry in which one subunit in each has been aligned (black arrow) shows thesimilarity of the structure of each subunit but a huge difference in subunit packing. The yellow surface has a one-starthelical symmetry with a twist of 221.0° and an axial rise of 11.7 Å. (Fig. 3e), while the cyan surface has C4 symmetry withan axial rise of 43.8 Å and a twist of 72.0° (Fig. 4e). The aligned subunit in the cyan structure is shown as mesh for clarity.(b and c) A helical net shows the lattice of subunits on the surface of a cylinder, using the standard convention that thecylindrical surface has been cut open and we are looking at the inside. (b) Two helical families are labeled in the helicalnet for the C4 helix. Within segments having this symmetry, the strongest observed connectivity between subunits isalong the left-handed four-start helices. (c) Three helical families are labeled in the helical net for the one-start helix. Aleft-handed one-start helix having ∼2.6 subunits per 30 Å pitch turn is labeled. The strongest observed connectivitybetween subunits within segments having a one-start symmetry is along the left-handed three-start helices and the right-handed two-start helices.
463The Structure of an Archaeal Pilus
power spectra from different bins appear to shiftcontinuously in position, with no indication of a fewdiscrete states.
Discussion
The putative proteins involved in pilus assembly inM.maripaludis have been identified as being similar tobacterial type IV pili in terms of having an N-terminal signal peptide and a predicted N-terminalhydrophobic α-helix.14 Subsequent genetic andbiochemical analysis (S.Y.M.N. et al., unpublishedresults) has confirmed the essential nature ofMMP0236 (epdB) and MMP0237 (epdC), two pilin-like genes from M. maripaludis, for pilus formation.The symmetry of bacterial type IV pili has beendetermined unambiguously only for the Neisseriagonorrhoeae pilus,24 in which subunits are related bya 10.5 Å rise and a 100.8° azimuthal rotation along aright-handed one-start helix. The N-terminal hydro-phobic α-helices in this bacterial type IV pilin packtogether to form a rather solid core, just as a similarpacking is observed in archaeal flagellar filamentscomposed of subunits that appear to be homologs ofbacterial type IV pilin.3,10
The two M. maripaludis pilin-like genes have theshort atypical signal peptide ending in a conservedglycine followed by a hydrophobic segment that istypical of type IV pilins. In addition, the signalpeptide is removed from EpdC by a type IV prepilinpeptidase homologue, EppA.14 Despite beingassembled from a subunit that has putative structuralhomology with bacterial type IV pilin, we show herethat the quaternary structure of the M. maripaludispilus is entirely different from both the bacterial typeIV pilus and the archaeal flagellar filament. Inaddition, we show that dramatic differences insubunit packing arrangements can exist, correspond-ing to two different helical symmetries, and theseappear to coexist within the same archaeal pilifilaments. An interesting question, one thatwe cannotanswer at this time, is whether the two differentsymmetries that we observe within these pili are dueto the presence of the two different pilin-like proteinsMMP0236 and MMP0237. This coexistence of twovery different symmetries is very similar to what hasrecently been reported for an archaeal Sulfolobusshibatae flagellar filament,4 with two distinct subunitpacking arrangements: one has helically arrangedstacked diskswithC3 symmetry, while the other has apure helical symmetry with∼3.3 subunits per turn of
464 The Structure of an Archaeal Pilus
a one-start helix. While for theM. maripaludis pili andthe S. shibatae flagellar filament the two symmetriescoexist, it has been known that within families ofclosely related filamentous phage two distinctlydifferent helical symmetries exist for different mem-bers of the family.25,26 The symmetry of the class Iparticles is a fivefold rotation axis combinedwith an ahelical rotation of ∼36° between adjacent rings ofsubunits, while the symmetry of the class II particlesinvolves ∼5.4 subunits per turn along a one-starthelix. It was suggested that despite this largedifference in helical symmetry, the protein packingmight be conserved in the two classes of filamentousbacteriophage.27 However, our results suggest thatprotein packing is not conserved between the twoforms of the M. maripaludis pilus and differentcontacts must occur between proteins within thesame filament.In contrast to the bacterial type IV pili that have a
rather solid core, in the Salmonella flagellar filamentthe N- and C-terminal α-helical D0 domains fromadjacent subunits are packed tightly together to forma hollow lumen that has a diameter of ∼20 Å.28 Thebacterial type III secretion system (T3SS) has beenknown to have homology with the bacterial flagellarsystem,29 and models for various components of theT3SS also involve packing of theseα-helices to form ahollow lumen.15,30 Here, we show that archaeal pilifilaments with either C4 symmetry or one-starthelical symmetry have a central lumen. In the caseof one-start helical symmetry, there is a ratheruniform central lumen that is slightly smaller (atthe available resolution) that what is seen in bacterialflagellar system and T3SS EspA filament. For the C4symmetry segments, the central lumen is muchmoremodulated. The naive expectation would be that thearchaeal pilus would be more similar to the bacterialtype IV pilus and the archaeal flagellar filament,neither of which have a hollow lumen. The possiblerole of such a lumen in secretion or assembly of thepilus at the distal end is intriguing. Perhaps archaeaassemble flagella by incorporation of subunits at thebase like bacterial type IV pili, while archaeal pili areassembled by incorporation of subunits at the distalend like bacterial flagella.While it is generally accepted that sequences
diverge more rapidly than structure over the courseof evolution,31,32 it has been suggested that quatern-ary structure may be more sensitive to small changesin sequence.33 For example, the overall fold ofeukaryotic actin and bacterial ParM has remainedrelatively similar over huge evolutionary distances,34
but the filaments formed by these proteins hasdiverged greatly.35 Dramatic changes in quaternarystructure, with only small changes in tertiary struc-ture, may play an important role in evolutionarydivergence. Despite being built from a subunitexpected to be similar to a bacterial type IV pilin, weshowhere that the quaternary structure of an archaealpilus is very different from both the bacterial type IVpilus and the archaeal flagellum (composed ofsubunits with homology to bacterial type IV pilin).What is more, distinctly different quaternary struc-
tures exist within the same archaeal pilus. This showsthat subunits must have the ability to switch betweenvery different contacts with their neighbors. Poly-morphic switchingwithin bacterial flagellar filamentshas been studied extensively,36 and involves quitesmall changes in the local packing of subunits thatchange the macroscopic properties of the flagellarfilaments. While we do not understand the functionsof these archaeal pili, understanding what regulatesthe polymorphic switching that they can undergowilldoubtless be important to understanding functionalmechanisms.
Materials and Methods
Purification of pili filaments from M. maripaludis ΔflaK
The purification of pili filaments was performed onM. maripaludis ΔflaK, which is a non-flagellated butpiliated strain. Cells (8 L) were grown for three days inBalch III medium at 37 °C under an atmosphere of CO2/H2(20:80)with shaking (110 rpm). The cellswere harvested bycentrifugation (5000g, 10 min) and resuspended in buffer(10 mM Tris–HCl, 2% NaCl, pH 7.0). Cell lysis wasobtained through the addition of the nonionic detergentOP-10 (Nikko Chemicals Co. Ltd., Tokyo, Japan) to a finalconcentration of 1% (v/v), with DNase/RNase added toreduce viscosity. The reaction was incubated at roomtemperature with occasional inverting for 90 min, afterwhich the sample was centrifuged at 2000g for 10 min. Theresulting supernatant was subjected to precipitation inpolyethylene glycol with the addition of 1 M NaCl, 20%(w/v) polyethylene glycol 8000 to a final concentration of10% (v/v) and kept on ice with shaking for 2 h. Pelletsobtained by centrifugation at 2000g for 10 min wereresuspended and subjected to banding in a KBr gradientfor further purification as described for flagella isolation.37
The diffuse white band containing the pili was removed,diluted with distilled water and pelleted by centrifugationto obtain the final pili sample.
Imaging and analysis
Microscopy was used for imaging: a Tecnai 12 transmis-sion electron microscope (TEM) for the negatively stainedsamples and a Tecnai F20 TEM with a field emission gunsource for the unstained frozen-hydrated samples. Thenegatively stained samples were applied to a carbon filmand stained with 2% (w/v) uranyl acetate. The samples forcryo-EM were applied to holey carbon (Quantifoil) orcontinuous carbon film on the EM grids. The micrographswere taken on film at a magnification of 30,000× (80 keV)for the negatively stained samples and a magnification of50,000× (200 keV) for the frozen-hydrated samples.Negatives were scanned with a Nikon Coolscan 8000 as16 bit images using a raster of 4.2 Å/pixel for the negativelystained samples and 2.4 Å/pixel for the frozen-hydratedsamples.The CTF was determined from each cryo-micrograph
(n=66) using either the carbon film surrounding the holeor the continuous carbon film support. The defocus valuesspanned the range from 1.7–3.5 μm. All cryo-EM imageswere multiplied by the theoretical CTF to correct for phasereversals and to optimize the signal-to-noise ratio. Finalreconstructions were then divided by the weighted sum of
465The Structure of an Archaeal Pilus
the squared CTF functions (as the images had beenmultiplied by a CTF twice: once by the EM, and once byus). For negatively stained samples, no CTF correctionwas necessary due to the fact that the first zero of the CTFwas beyond the resolution of the reconstruction.We extracted 28,577 overlapping segments, each 200
pixels or 480 Å long, from images of the frozen-hydratedsamples. All these segments were down-sampled to4.8 Å/pixel for data analysis and 3D reconstruction toexclude high-resolution information dominated by noise.We determined that the resolution of the final reconstruc-tions was not limited by the coarser sampling. The shiftbetween adjacent boxes was ten pixels in all cases. Thislarge overlap improves the signal-to-noise in the recon-struction, as adjacent segments correspond to projectionsof the structure from different azimuthal angles.
Mass per unit length measurements from STEM
Mass per unit length measurements from STEM weremade by similar methods as described.38 Freeze-driedspecimens were prepared according to the standardmethod of the Brookhaven STEM facility†. Digital dark-field micrographs of freeze-dried specimens wererecorded with 512×512 pixels at raster steps of 1.0 nm or2.0 nm per pixel using STEM from the Brookhaven STEM.The images were analyzed using the PCMass program(available from the Brookhaven STEM resource). Theresulting data were normalized to the known mass perunit length of tobacco mosaic virus (131.4 kDa/nm).Histograms were calculated with 1 kDa/nm bins. AGaussian was then fit to the distribution using the Originsoftware package (OriginLab company).
Acknowledgements
This work was supported by NIH EB001567 (toE.H.E.) and a Discovery Grant from the NaturalSciences and Engineering Research Council ofCanada (to K.F.J.). We thank Martha Simon of theBrookhaven STEM facility for imaging our samples.
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