A comparison of liquid nitrogen and liquid helium as...

Journal of Structural Biology 153 (2006) 231–240

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A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography

Cristina V. Iancu, Elizabeth R. Wright, J. Bernard Heymann 1, Grant J. Jensen ¤

Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA

Received 29 June 2005; received in revised form 16 November 2005; accepted 7 December 2005Available online 4 January 2006

Abstract

The principal resolution limitation in electron cryomicroscopy of frozen-hydrated biological samples is radiation damage. It has longbeen hoped that cooling such samples to just a few kelvins with liquid helium would slow this damage and allow statistically better-deWned images to be recorded. A new “G2 Polara” microscope from FEI Company was used to image various biological samples cooledby either liquid nitrogen or liquid helium to »82 or »12 K, respectively, and the results were compared with particular interest in thedoses (10–200 e¡/Å2) and resolutions (3–8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrastat »12 K through the Wrst several tens of e¡/Å2, after which small bubbles appeared. Single particle reconstructions from each image in adose series showed no diVerence in the preservation of medium-resolution (3–5 nm) structural detail at the two temperatures. Tomo-graphic reconstructions produced with total doses between 10 and 350 e¡/Å2 showed better results at »82 K than »12 K for every dosetested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography. 2006 Elsevier Inc. All rights reserved.

Keywords: Electron cryomicroscopy; Tomography; Helium cooling; Radiation damage; CryoEM

1. Introduction

Biological materials can be imaged in transmission elec-tron microscopes in a life-like, “frozen-hydrated” statethrough the use of specialized cryostages that keep samplesfrozen while they are inside the microscope column. Forthese samples, radiation damage is the principal resolutionlimitation, far exceeding others such as electron optical per-formance. The Wrst cryostages cooled samples to »90 Kthrough thermal contact with liquid nitrogen. After it wasobserved that radiation damage proceeded much moreslowly at low temperature, it was hoped that additionaldose resilience might be realized through further cooling(Glaeser, 1971). New microscopes were engineered to cool

* Corresponding author. Fax: +1 626 395 5730.E-mail address: [email protected] (G.J. Jensen).

1 Present address: Laboratory of Structural Biology Research, NationalInstitute of Arthritis, Musculoskeletal and Skin Diseases, National Insti-tutes of Health, Bethesda, MD 20892, USA.

1047-8477/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2005.12.004

specimens to just a few kelvins with liquid helium, but earlystudies were highly variable and “unable to Wnd deWniteevidence that there is an improvement in radiation resis-tance on going from liquid nitrogen to liquid helium”(International Study Group, 1986). More recently, how-ever, Stark et al. found that the so-called “cryoprotectionfactor” at 4 K (under liquid helium cooling) was 1.4 and 2.5times better than at 98 K (under liquid nitrogen cooling) for7 and 3 Å spacings, respectively, in two-dimensional proteincrystals (Stark et al., 1996). Part of the ambiguity in pastwork stemmed from the fact that previous microscopesused either liquid nitrogen or liquid helium exclusively, sothat comparisons had to be made between diVerent micro-scopes in diVerent labs or settings.

All previous reports have focused on the doses (»10 e¡/Å2 or less) and resolutions (»7 Å or better) of interest toelectron crystallography, and used the same basic measure-ment—the fading of crystal diVraction patterns. We wish toemphasize that the question of whether liquid helium cool-ing is actually advantageous or not depends, of course, on

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mailto: [email protected]

232 C.V. Iancu et al. / Journal of Structural Biology 153 (2006) 231–240

the type of sample and goals of the project. For two-dimen-sional crystallographic studies probing near-atomic resolu-tion, for instance, specimen atoms must remain in theiroriginal locations, and doses of just a few e¡/Å2 are typi-cally used. For single particle analysis studies seeking toresolve secondary structure, structures the size of � helicesand � sheets must remain largely intact, and doses of »10–20 e¡/Å2 are standard. Finally, for tomographic studiesseeking to visualize shapes of individual proteins, radiolyticfragments of protein domains must remain only approxi-mately in place, and doses of »20 to 200 e¡/Å2 are common.Furthermore, while lower temperatures may or may nothelp reach any of these goals, potential disadvantages suchas increased charging or drift must also be considered. Thussuccesses or failures with liquid helium cooling in one con-text are not necessarily predictive in others.

Here, our focus is tomography. We compare liquidnitrogen and liquid helium cooling with methods that arediVerent from all previous work in the nature of the sample,the doses used, the type of data collected, the measures ofquality, and the degree of control. Whole cells and individ-ual, large protein complexes are imaged through doses of10–350 e¡/Å2. Image quality is measured both qualitativelyand quantitatively in the resolution range of 3–6 nm. Theinstrument used was one of the new series of “Polara”microscopes from FEI, which allows liquid nitrogen andhelium to be exchanged repeatedly, all while a single grid isbeing imaged in a single microscope, without other con-founding variables. Disappointingly, in this context we Wndliquid helium cooling to be disadvantageous. In the com-panion paper, we report several additional observationsthat help explain this result (Wright et al., companionpaper). After conWrming that vitreous ice collapses from alow to a high density state when irradiated at »12 K, we goon to show that the collapse alone is not the immediateproblem. Instead, we speculate that the key issue is howradiolytic fragments aggregate within the high density ice.

2. Results

A 300 keV “G2 Polara” transmission electron micro-scope from the FEI Company was installed in our lab at theCalifornia Institute of Technology in the fall of 2003. It isequipped with a new cartridge-based sample holder speciW-cally designed to allow liquid helium cooling. An externaldewar system is permanently mounted to the side of thecolumn consisting of an inner “helium” dewar surroundedby an outer “nitrogen” dewar. The inner dewar is thermallyconnected to the tip of the stage, so that when a cartridge isthreaded onto the stage, it is cooled to near the temperatureof the cryogen in the inner dewar within just a few minutes.The Polara design is diVerent from some previous liquidhelium cooled microscopes in that there is no specialrequirement for helium per se—the inner dewar can beWlled with either liquid helium or nitrogen without compli-cation, and in fact the cryogen can be exchanged backand forth all while a single cryosample is being imaged.

The temperature of the sample when liquid helium is pres-ent in the inner dewar was estimated by mounting a silicondiode thermometer on a special cartridge while the columnwas open in the factory. For our microscope, that tempera-ture was measured to be 11.5 K. During operation, a ther-mocouple provides real-time measurements of thetemperature of the “cryobox” immediately surroundingand in thermal contact with the cartridge. When the innerdewar is Wlled with liquid helium, this reads 10 K. Whencooled with liquid nitrogen, it reads 82 K.

To begin our studies, simple dose series of several fro-zen-hydrated biological samples were recorded at the twotemperatures, and the radiation damage trajectories wereobserved. The Wrst sample tested was intact, frozen-hydrated cells of a small bacterium, Mesoplasma Xorum.Cells were plunge frozen, inserted into the microscope, andcooled to »82 K with liquid nitrogen. A suitable cell wascentered under the beam with less than 1 e¡/Å2 dose, andthen approximately 70 exposures were recorded with 10 e¡/Å2 each. Several key images are shown in the Wrst columnof Fig. 1. Next the grid was retracted into the liquid nitro-gen cooled “multispecimen transfer system,” which resideswithin the column vacuum, and the liquid nitrogen in theinner dewar was replaced with liquid helium. After the tem-perature of the sample area settled to »12 K, the grid wasre-threaded onto the stage, allowed to cool (to »12 K), andanother Me. Xorum cell several microns away from the Wrstwas centered under the beam. An identical dose series wasrecorded (Fig. 1, second column).

At »82 K, the contrast from the cell membrane and mac-romolecular complexes inside the cell appeared qualita-tively similar throughout the dose series, but did spread andsmear gradually. In the Wrst image, the cell was punctuatedwith small and distinct densities, which are the projectionsof internal macromolecules, and the membrane appeared asa dark, single band. In subsequent images, the internal den-sities became less distinct and slightly rearranged. Never-theless, densities the size of single large protein complexescould easily be traced through the images (circles in Fig. 1column 1), suggesting that their basic shapes were pre-served, until small “bubbles” appeared after »160 e¡/Å2

and the internal structures were catastrophically perturbed.At »12 K, the Wrst image was qualitatively indistinguish-

able from the Wrst image at »82 K, but in subsequentimages the membrane contrast faded quickly. The mem-brane became largely invisible after »40 e¡/Å2, and thensplit into two dark bands separated by a light interior band.This trilaminar structure had a slightly larger total widththan the original membrane when it Wrst appeared (»80 e¡/Å2), and then expanded non-uniformly as the dose seriescontinued. At the end (700 e¡/Å2), large bubbles appearedbetween the dark outer layers (not shown). The relativecontrast between the layers increased steadily with dose.Concerning the appearance of internal macromolecularcomplexes, they seemed to smear and rearrange slightlywith dose just as those imaged at »82 K, until again smallbubbles disrupted their structure grossly after »160 e¡/Å2.

C.V. Iancu et al. / Journal of Structural Biology 153 (2006) 231–240 233

250 nm.)

Fig. 1. Dose series of intact, frozen-hydrated bacterial cells. Me. Xorum cells were plunge frozen and imaged in the Polara electron cryomicroscope. Seventyimages were recorded of each cell with 10 e¡/Å2/image. The Wrst and second columns show images recorded at »82 and »12 K, using liquid nitrogen andliquid helium as cryogen, respectively. One particular cluster of density is circled in each image of the Wrst column to facilitate comparison. The third andfourth columns show images recorded again at »12 K, but where the sample was brieXy warmed up in the liquid nitrogen cooled multispecimen holder, asif for rotation in the Xip-Xop rotation stage, and then re-cooled in the column to »12 K. In the third column, the sample was warmed once after a cumula-tive dose of 30 e¡/Å2. In the fourth column the sample was warmed and re-cooled after every 30 e¡/Å2. Note how the membrane contrast fades and is thenreplaced by bubbles at »12 K but not »82 K. This eVect is delayed by one warming cycle, and prevented indeWnitely by iterative warming cycles. (Scale bar


Fig. 2. Dose series of liposomes. Dose series were recorded as in Fig. 1, but this time of liposomes. Pure lipids exhibit the same contrast eVects as the cellmembrane, but at approximately twice the dose. While bubbles form on the carbon support at both temperatures, they are much larger and coalesce to agreater extent at »82 K than »12 K. Bubbling on the carbon is apparently prevented by iterative warming cycles. (Scale bar 200 nm.)


Fig. 3. Dose series of puriWed protein complexes. Dose series of a puriWed protein complex, hemocyanin, are shown. At »82 K, the proteins becomesmeared as their Wne structure is destroyed, but their general shapes are still evident even after 300 e¡/Å2. As for lipids, at »12 K the contrast from puriWedprotein fades and is eventually replaced by small bubbles. A single warming cycle delays the eVect slightly, but iterative warming cycles prevent it indeW-

nitely. (Scale bar 200 nm.)


Fig. 4. Information loss as a function of dose at »82 and »12 K. From thehemocyanin dose series, 128 particles were chosen and used to produceindependent single particle reconstructions from each image. As a control,an equivalent number of arbitrary positions not containing particles werealigned and merged, yielding the resolution labeled as “noise alone.” Therate of information loss at »82 and »12 K was assessed by tracking theresolution of the reconstructions. Two curves for each temperature areshown. The notable diVerences were that the Wrst image at »12 K wasoften blurred, and essentially no useful information was recovered fromthe images at »12 K after about 150 e¡/Å2.

Unlike the cellular components imaged at »82 K, however,their contrast faded noticeably and the nature of the bub-bling was diVerent in that the bubbles were more numerousbut smaller, and did not coalesce as quickly.

Our Polara was recently equipped with the prototype“Xip-Xop” rotation stage, which allows grids to be rotated90° within the column vacuum to enable dual-axis cryoto-mography (Iancu et al., 2005). As designed, it had onemajor potential liability, which was that the grid is rotatedin the so-called multispecimen transfer system, which canonly be liquid nitrogen cooled. Thus, if a Wrst tilt-series wererecorded at »12 K, before the second, orthogonal tilt-seriescould be recorded, the grid would have to be warmed up to»82 K for rotation and then re-cooled to »12 K. If liquidhelium cooling did in fact constrain radiation damage ashoped, such warming might release those constraints beforethe second tilt-series. To investigate this possibility, werecorded dose series at »12 K wherein the samples wereretracted into the multispecimen holder as if for rotation(and thus warmed to »82 K and re-cooled to »12 K, here-after referred to as a “brief warmup/cooldown cycle”) after30 e¡/Å2 to mimic collection of a typical dual-axis tilt-series

Fig. 5. Tomogram quality as a function of dose at »82 and »12 K. Full tilt-series were recorded and tomographic reconstructions calculated of Welds offrozen-hydrated hemocyanin using total doses from 10 to 350 e¡/Å2 at both »82 and »12 K. From each tomogram, 100 hemocyanin particles were chosenand compared to a known higher resolution structure by cross correlation. The overall quality of each tomogram was then assessed by calculating the aver-age cross-correlation coeYcient (plotted) and the standard deviation (shown as half error bars for clarity). As a control, an equivalent number of arbitrarypositions not containing particles were also selected and analyzed (labeled as ‘noise’). Above or below each data point, inset panels show isosurface rendi-tions at 2.5 standard deviations above the mean of the best reconstructed particle after denoising from “top” and “side” views. At every dose, the results at
»82 K were superior. For scale, hemocyanin is barrel-shaped, »30 nm in diameter and »35 nm long.


(Fig. 1, column 3). Surprisingly, the radiation damage tra-jectory seemed just like that seen at »12 K without anywarming, except delayed by approximately 30 e¡/Å2. Spe-ciWcally, the Wrst image after warming looked like the origi-nal, but the loss and subsequent splitting of the membranecontrast then followed. This prompted us to test a fourthprotocol, wherein the sample was iteratively retracted andreplaced (warmed to »82 K and then re-cooled) every 30 e¡/Å2 (Fig. 1, column 4). In this case the appearance of themembrane remains unchanged, and the results are likethose recorded at a constant »82 K (column 1) except thatinternal bubbling is further delayed.

To investigate whether these membrane eVects were dueto integral membrane proteins or the cellular environment,we recorded similar dose series of pure liposomes (Fig. 2).At »82 K, there was no visible change or bubbling in thelipid itself, even to the end of the dose series (800 e¡/Å2).The same loss of membrane contrast observed at »12 Kwas seen again here, followed by splitting into a trilaminarstructure, but importantly, the eVect came later in the doseseries. While it is diYcult to assign speciWc numbers, thefading and splitting occurred at about twice the doserequired for the analogous eVect in the Me. Xorum cellmembrane (note the range of doses in Fig. 2 is nearly dou-ble that shown in Fig. 1). Brief warmup/cooldown cycleshad the same qualitative eVect as seen before in the cell. Theother interesting phenomenon in these dose series is thenature of the “bubbling” that occurs on the carbon sup-port. At »82 K, bubbles Wrst became visible at »50 e¡/Å2,and then grew and coalesced. At »12 K, however, bubblingwas delayed, and the bubbles appeared much smaller andremained distinct. Surprisingly, while the bubbles on thecarbon were unaVected by a single warming cycle, iterativewarming cycles prevented visible bubbling, even through600 e¡/Å2.

Next we recorded analogous dose series of several large,puriWed protein complexes including hemocyanin, the 20 Sproteasome, and the pyruvate dehydrogenase multienzymecomplex. Images from the hemocyanin dose series areshown in Fig. 3. At »82 K, the contrast from individualhemocyanin molecules gradually smeared through the Wrstseveral hundred e¡/Å2, but did not fade, and the grossshapes of the molecules remained evident through morethan 300 e¡/Å2 (column 1). Bubbling was only observedwhere the proteins were aggregated. At »12 K, the Wrstimage was quite similar to the Wrst image at »82 K, butthereafter the contrast of the proteins gradually faded, untilthey were virtually invisible at »200 e¡/Å2. Past 200 e¡/Å2,small bubbles appeared in a strikingly similar pattern as thedomains of the original proteins, and then grew with addi-tional dose (column 2). The pattern of contrast fading at»12 K was not visibly diVerent when the sample was brieXywarmed and cooled just once after 30 e¡/Å2 (column 3), butwhen the sample was taken through iterative warmup/cool-down cycles (column 4), the contrast was actually pre-served, resembling at each dose the series recorded at»82 K. The 20S proteasome and pyruvate dehydrogenase

multienzyme complexes manifest the same patterns in con-trast fading and bubbling as hemocyanin, as did hemocya-nin in thick ice (data not shown).

To compare the usefulness of each image quantitativelyas a function of dose, three-dimensional hemocyanin recon-structions were calculated from the particles found in eachimage with standard “single particle” averaging methods,using the known higher resolution model (Mouche et al.,2003) of hemocyanin as the reference for alignment and res-olution assessment (Fig. 4). Two dose series having thesame number of particles, defocus, and pixel size were ana-lyzed each for both »82 and »12 K. To assess the inXuenceof reference bias, an equal number of ice regions not con-taining particles were also chosen, aligned to the known ref-erence, and merged to form a three-dimensionalreconstruction. This reconstruction of noise was also com-pared to the reference through Fourier shell correlation fol-lowing identical procedures, yielding an apparent“resolution” of 4.3 nm. Thus, two factors contribute to res-olution here: the true information about particle shapepresent in the images, and aligned noise. To track the pres-ervation of useful information and reduce the impact ofaligned noise, the orientation angles of each particle wereestimated from the Wrst (or second in the case of »12 K)image and then Wxed for the remainder of the dose series.Only particle positions were reWned in subsequent images.The results were highly reproducible, and showed nodetectable diVerence in the cryoprotection factor for dosesfrom 10–120 e¡/Å2 in the 3.5–4.5 nm resolution range. After120 e¡/Å2, however, the resolutions from the helium cooledseries degraded rapidly. This was expected, since the parti-cles are essentially invisible. It was also observed that thevery Wrst image recorded at »12 K was frequently blurred,as evidenced by its anomalously poor resolution. Note thatimages of the same 128 physical particles were used toproduce each reconstruction. The experiment was repeatedwith various defocus values and pixel sizes, all with thesame result. The same result was also obtained when theorientation for each particle was taken from the image cor-responding to its highest Wgure-of-merit in the alignmentroutine, rather than the Wrst or second of the series.

To complete the comparison of cryogens in the full con-text of tomography, tilt-series were recorded of Welds of fro-zen-hydrated hemocyanin with diVerent total dosesbetween 10 and 350 e¡/Å2 at both temperatures. One hun-dred particles from each tomogram were selected andaligned in three dimensions to the known 12 Å structure(Mouche et al., 2003). The average cross-correlation coeY-cient between the model and the reconstructed particles wasplotted as a function of total dose used for the tilt-series(Fig. 5). All other variables besides temperature and totaldose were kept as similar as possible, including defocus,magniWcation, ice thickness, tilt range, and tilt step. Whileimages could be aligned and produced meaningful recon-structions with as few as 10 e¡/Å2, the results improved withdose for both temperatures up to 120 e¡/Å2, presumablydue to reduction of shot noise. Past 120 e¡/Å2, however, the


results began to deteriorate, presumably because of radia-tion damage. At all doses, the results at »82 K were betterthan those at »12 K, and the diVerence became more pro-nounced at higher doses, probably due to lost contrast inthe later images at »12 K.

3. Discussion

Through dose series and tomograms of various frozen-hydrated samples including intact cells, liposomes, and sev-eral puriWed protein complexes, we have shown empiricallythat cooling samples with liquid helium rather than liquidnitrogen is unexpectedly disadvantageous. The mostimportant observation was that at »12 K, the contrast ofproteins and lipids faded steadily with irradiation in theWrst several tens of e¡/Å2, and was then replaced by high-contrast but artifactual features such as trilaminar struc-tures in the place of membranes and small bubbles in theplace of protein domains.

Two types of quantitative measures were used to detectany diVerence in the cryoprotection factors at »82 and»12 K. In the Wrst, independent single particle reconstruc-tions of hemocyanin were calculated from individualimages within a dose series. While the resolutions steadilydecreased with dose as expected, no diVerence was seen inthe rate of this decrease at the two temperatures. Instead,the only signiWcant diVerences were (1) that the Wrst imagerecorded at »12 K was frequently blurred, and (2) that after»120 e¡/Å2, only the nitrogen cooled samples retained use-ful information about the shapes of the proteins. The sec-ond quantitative measure compared full tomographicreconstructions of hemocyanin at diVerent total doses atthe two temperatures. At every total dose from 10 to 350 e¡/Å2, the results were better when liquid nitrogen was usedthan liquid helium. The best reconstructions of all, as mea-sured by cross-correlation with the known higher-resolu-tion structure, were obtained with 120 e¡/Å2 and liquidnitrogen cooling.

While the conclusion that liquid helium cooling wasinferior in these comparisons was clear, its explanation wasnot. After observing the gradual loss of contrast at »12 K,our Wrst thought was that the ice was collapsing and simply“contrast-matching” the proteins and lipids. As reported inthe companion paper, however, extensive additional experi-ments were performed to understand the behavior of ice atthe two temperatures (Wright et al., companion paper). Thekey results were that, in conWrmation of the existing litera-ture (Heide, 1984), (1) vitreous ice does indeed collapse intoa higher density phase when irradiated at »12 K, (2) thishappens rapidly–within just the Wrst 2–3 e¡/Å2, and (3) thehigh density ice spontaneously expands back to the lowdensity state when warmed to »82 K over a period of atleast several minutes. Thus collapse of the ice cannot aloneexplain the loss in contrast seen here for two reasons. First,the rates of the two processes are diVerent by an order ofmagnitude (two versus several tens of e¡/Å2). Second, theloss of contrast proceeded at diVerent rates for diVerent lip-

ids and diVerent proteins, even though they were in similaror in some cases identical buVers which should changephases at the same rate.

Instead, we speculate that the diVerences in contrast andbubbling at »82 and »12 K are due to diVerences in howwater molecules and radiolytic fragments diVuse in the twoice phases. Various small molecules are expected to formwhen frozen-hydrated organic material is irradiated,including hydroxyl radicals, hydrogen atoms, hydrogen gas,carbon monoxide, and carbon dioxide. There are reportsand/or claims in the literature that the mobility of each ofthese speciWc small molecules changes signiWcantly in thetemperature range of 4–90 K (see, for example, Glaeseret al., 1971; Heide, 1982, 1984; Jenniskens and Blake, 1994;Jenniskens et al., 1995; Sandford and Allamandola,1993a,b). We can still only speculate, however, exactly howsuch small molecules behave in the unknown local condi-tions surrounding a protein, for instance, as it is beingdestroyed by irradiation, cooled by a cryogen but locallyheated by inelastic scattering events, and embedded in vit-reous ices of variable composition (i.e. the diVerent buVersand cell culture medias were used here). Furthermore,diVerent methods and conditions of formation seem to pro-duce diVerent high density vitreous ices (Angell, 2004), so itis unclear which mobility studies are applicable in the pres-ent context and which are not. Finally, surprises such as theWnding that acoustic waves in room-temperature liquidwater can produce small bubbles with local temperatures ofmillions of kelvins (Crum and Matula, 1997) highlightshow misguided simple intuition can be!

Nevertheless, two facts seem clear: (1) when covalentbonds are broken by the beam, the newly unbonded atomswill repel each other strongly through van der Waals and/orCoulombic forces, and (2) the low density state of vitreousice must have larger or more intermolecular spaces than thehigh density state (Jenniskens et al., 1995). At »82 K then,radiolytic fragments and byproducts (like hydrogen gas)likely diVuse away from each other gradually through theseintermolecular spaces. The surrounding water and otherfragments might rearrange slightly, as energy deposited bythe beam dissipates, and the net eVect in the image wouldbe an apparent “smearing.” If the diVusion of small radio-lytic fragments and byproducts is blocked, however, bysome denser material like proteins in a crowded cytoplasm,a carbon support, or even just the denser vitreous ice at»12 K, the accumulating repulsion of radiolytic fragmentsmight eventually force open pockets of relatively lowerdensity material, described here as “bubbles.” Thus, thebubbles are likely to contain hydrogen gas and other smallmolecules. The formation and incremental growth of thesebubbles is likely responsible for the gradual loss of contrastseen. We also observed that when samples at »12 K areirradiated and then brieXy warmed and cooled, as forinstance is required by the Xip-Xop cryorotation holder,bubbling is prevented and normal contrast is retained. Ourinterpretation is that while these cycles are not long enoughto cause global expansion of the ice into its low density


form (Wright et al., companion paper), they are suYcient torelieve local strains like the forces that would otherwiseresult in bubble formation.

4. Materials and methods

4.1. Dose calibration

All reported doses were measured using the 2048 £ 2048pixel CCD camera of our Gatan (Pleasanton, CA) ImagingFilter. The number of counts reported by this camera perprimary electron was exhaustively calibrated using fourindependent ammeters: an in-house Faraday cup; theGIF’s drift tube; the small, insertable Xuorescent screen;and the large Xuorescent viewing screen. First, an in-houseFaraday cup was installed at the level of the viewing screenand connected to an external ammeter in such as way that acondensed beam could be deXected either to an exposedpart of the viewing screen or into the Faraday cup. The cur-rent to either of the two Xuorescent screens is reported bythe FluScreenMgr panel of the Feispy application based onfactory calibrations. The measurements from the twoscreens were in all cases essentially identical, and these werefound to agree well with measurements taken with the Far-aday cup, varying predictably by up to 30% (the worst case)across two orders of magnitude in intensity (0.034–1.980 nA) and at voltages of 120, 200, and 300 keV. Afterthe Faraday cup was removed, beam currents were againmeasured by deXecting the entire beam into the GIF drifttube and recording its output current with an externalammeter. These measurements were all »20% higher thanthose from the viewing screens and the Faraday cup. TheFaraday cup was judged to be the most accurate of the fourmethods, and was used to calibrate the sensitivity of theGIF CCD camera as producing 7 counts/primary electron.This was done by condensing the beam entirely within theCCD, exposing for exactly 1 s, integrating the total countsreported on the CCD, and dividing by the total doseapplied. The sensitivity of the CCD was conWrmed to belinear throughout the range used in this study.

4.2. Samples

Mesoplasma Xorum was obtained from the ATCC(strain #33453) and cultured in Mycoplasma medium(ATCC medium #243) with the addition of phenol red asa growth indicator. Cells were allowed to reach late expo-nential phase at which time they were centrifuged at10,000g for 3 min and resuspended in growth media. Lip-osomes were formed from either pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a 1:1 mixture ofDPPC and 1,2-dioleoyl-sn-glycero-phosphoethanol-amine-N-[4-(p-maleimidophenyl) butyramide] (MPB PE)by extrusion through 0.2 �m Wlters. PuriWed Escherichiacoli pyruvate dehydrogenase complex, Megathura crenu-lata Keyhole Limpet hemocyanin (A.G. ScientiWc), andMethanosarcina thermophila 20S proteasome (Calbio-

chem) were diluted in 50 mM sodium phosphate, pH 7.2to Wnal protein concentrations of 2, 8, and 3 mg/ml,respectively.

Ten-nanometer colloidal gold (Ted Pella) was combinedwith the samples and also applied to the grids separately.Protein solutions and cell suspensions were Xash frozenonto R1.2/1.3 carbon grids (Quantifoil) in liquid ethaneusing a Vitrobot (FEI, The Netherlands) and the followingtypical conditions: 100% humidity, 23 °C, 4�l/grid if manu-ally applied, 2.0–3.5 s blot time, ¡2 to ¡3 mm blot oVset.Grids with ice thickness and gold distribution suitable fordata collection were reproducibly generated using theseconditions.

4.3. Dose series protocols

All images were energy Wltered (20 eV slit width) andrecorded on the 2048 £ 2048 CCD of the Polara TEM’sGIF. Successive images were collected at 30 s intervals; at adose/image of 5 e¡/Å2 (hemocyanin) or 10 e¡/Å2 (Me.Xorum; liposomes; mixture of hemocyanin, 20S proteasomeand pyruvate dehydrogenase); at 6.7 Å/pixel; using defocusvalues of ¡6 �m (hemocyanin), ¡8 �m (liposomes), or¡15 �m (Me. Xorum) (Wrst zeroes of the contrast transferfunction at 1/3.4, 1/4.0, and 1/5.4 nm¡1, respectively); eitherat »82 or »12 K. Typically, the areas imaged at the twotemperatures came from the same grid square and wereseparated by »10 �m to ensure similarity in ice thickness,sample, and gold distribution.

4.4. Single-particle reconstructions

For Fig. 4 the hemocyanin dose series were taken with10 e¡/Å2, a defocus of ¡4 �m, and a pixel size of 6.7 Å. Theexperiment was repeated with a diVerent defocus value(¡6 �m), and a third time with a diVerent pixel size (9.8 Å),all with the same result. All processing steps were carriedout using the Bsoft (Heymann, 2001) and Peach (Leonget al., 2005) packages. For each hemocyanin dose seriesanalyzed, the images were mutually aligned, and then a setof 128 particles within the Quantifoil hole was selected andboxed out separately from each image in the series. Theposition and orientation of each particle were found bycomparison to projections of the published structure (Mou-che et al., 2003). Independent, three-dimensional recon-structions for each image in the series were then calculatedby weighted back-projection using reWned positions foreach particle image. Particle orientations for the data at»12 K were Wxed at the values obtained from the secondimage in the series, since the Wrst image was typicallysmeared. The resolution of each reconstruction wasassessed by the Fourier shell correlation method using athreshold of 0.5. An equal number of arbitrary positionsnot containing particles were treated identically to producean “aligned-noise-only” reconstruction as a control toassess reference bias. The resolution of the aligned-noise-only reconstructions varied consistently with the number of


noise “particles” used as expected. For 128 particles, the“resolution” was 4.25 nm.

4.5. Hemocyanin tomograms

Energy-Wltered (20 eV slit width) image tilt-series withtotal doses of 10, 25, 50, 80, 120, 200, and 350e¡/Å2 wererecorded from ¡69° to 69° with 3° step sizes, a defocus of¡6�m (Wrst zero of CTF at 1/3.4 nm¡1), and a pixel size of9.8 Å using the “predictive” UCSF tomography package(Zheng et al., 2004). All holes imaged at both temperatureswere chosen from one grid square on one grid with similarice thickness except one (80 e¡/Å2 at »12 K), which had to berepeated on a diVerent day and grid due to technicaldiYculties. Tomographic reconstructions were calculatedusing IMOD (Mastronarde, 1997). One hundred particlesfrom each tomogram were manually selected and aligned tothe published structure (Mouche et al., 2003) using the Bsoftpackage (Heymann, 2001) and the Peach distributed compu-tation system (Leong et al., 2005). The highest cross-correla-tion coeYcient, which identiWes the best three-dimensionalalignment, was used as a quantitative measure of reconstruc-tion quality. An equal number of arbitrary positions not con-taining particles within the tomograms were picked andaligned using identical calculations to assess the cross-corre-lation coeYcients from “noise.” For the surface renditioninsets in Fig. 5, particles were denoised with 10 cycles of non-linear anisotropic diVusion (Frangakis and Hegerl, 2001).

Acknowledgments

We thank Y. He for providing liposomes; T. Wagenkn-echt and J. Berkowitz for pyruvate dehydrogenase; J. Ben-jamin, P. Leong, and J. Ding for help with data collectionand image processing; and W. Tivol for reading the manu-script. This work was supported in part by NIH Grant PO1GM66521 to G.J.J., DOE Grant DE-FG02-04ER63785 toG.J.J., a Searle Scholar Award to G.J.J., the Beckman Insti-tute at Caltech, and gifts to Caltech from the Ralph M. Par-sons Foundation, the Agouron Institute, and the Gordonand Betty Moore Foundation.

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