Monolayer purification: A rapid method for isolatingprotein complexes for single-particleelectron microscopyDeborah F. Kelly, Danijela Dukovski, and Thomas Walz*

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115

Communicated by Stephen C. Harrison, Harvard Medical School, Boston, MA, January 28, 2008 (received for review December 7, 2007)

Visualizing macromolecular complexes by single-particle electronmicroscopy (EM) entails stringent biochemical purification, specimenpreparation, low-dose imaging, and 3D image reconstruction. Here,we introduce the ‘‘monolayer purification’’ method, which employsnickel-nitrilotriacetic acid (Ni-NTA) functionalized lipids for simulta-neously purifying His-tagged complexes directly from cell lysateswhile producing specimens suitable for single-particle EM. Themethod was established by using monolayers containing Ni-NTA lipidto specifically adsorb His-tagged transferrin–transferrin receptor(Tf-TfR) complexes from insect and mammalian cell extracts. Thespecificity and sensitivity of the method could be improved by addingimidazole to the extracts. The monolayer-purified Tf-TfR samplescould be vitrified and used to calculate a 3D reconstruction of thecomplex. Monolayer purification was then used to rapidly isolateribosomal complexes from bacteria by overexpressing a single His-tagged ribosomal subunit. The resulting monolayer samples allowedcalculation of a cryo-EM 3D reconstruction of the Escherichia coli 50Sribosomal subunit.

lipid monolayer � affinity purification � cryoelectron microscopy

Pure preparations are essential for structural studies of proteinsand macromolecular complexes. To simplify biochemical puri-

fication, recombinant proteins often are expressed with an affinitytag. One of the most widely used tags is the His tag, which consistsof six or more contiguous histidine residues. His-tagged proteinsexhibit high affinity [KD of �10�13 M at pH 8.0 (1)] for nickel-nitrilotriacetic acid (Ni-NTA) groups, which can be covalentlyattached to a separation matrix (2) and thus used for efficientprotein purification using metal ion affinity chromatography (3). Topurify macromolecular complexes, it is often sufficient to His-tagonly a single component of the complexes.

Lipid monolayers have been introduced as a method in electronmicroscopy (EM) to grow 2D protein crystals at the air–waterinterface (4). By including lipids in the monolayer with a derivatizedNi-NTA head group (referred to as Ni-NTA monolayers from hereon), the technique subsequently was used to grow 2D crystals ofHis-tagged proteins (5), which later proved suitable for calculating3D reconstructions by electron crystallography of vitrified samples(6). In an alternative approach, lipid monolayers also have beenused to prepare macromolecular complexes for structural studies bysingle-particle EM. For example, purified mammalian spliceosomalcomplexes were adsorbed to positively charged lipid monolayers,which could then be frozen and imaged by cryo-EM (7, 8).

In this work, we introduce an application of Ni-NTA monolayers,which we named ‘‘monolayer purification.’’ This technique usesNi-NTA monolayers to prepare His-tagged complexes for single-particle EM without prior chromatographic purification. Themethod was established and characterized by using as a testspecimen a His-tagged transferrin receptor (TfR) ectodomainconstruct bound to diferric transferrin (Tf). Once the experimentalparameters were determined, we used monolayer purification toisolate ribosomal complexes directly from extracts of Escherichiacoli cells expressing a His-tagged subunit of the human 60S subunit.The monolayer samples were vitrified and imaged with cryo-EM,

and the images were used to calculate a 3D reconstruction of theE. coli 50S subunit. Our studies thus establish the Ni-NTA mono-layer system as a means for rapidly purifying His-tagged macro-molecular complexes. A 25-�l sample, containing as little as 0.4�g/ml of His-tagged protein, is sufficient to produce a specimensuitable for structure determination by single-particle EM.

ResultsAdsorption of His-Tagged Tf-TfR Complex to Ni-NTA Monolayers. Weused the Tf-TfR complex to assess how efficiently Ni-NTA mono-layers can recruit His-tagged proteins to the lipid surface. Our TfRconstruct (residues 89–790) lacks the transmembrane and cyto-plasmic domains but contains a 6�His tag at the N terminus of thestalk, where it would connect to the transmembrane domain.Aliquots (25 �l) of the purified Tf-TfR complex (0.01 mg/ml in 50mM Hepes, pH 7.4, and 150 mM NaCl) were placed into wells ina Teflon block (Fig. 1a, step 1) and overlaid with a neutral1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) lipid mono-layer spiked with increasing amounts of Ni-NTA lipid (Fig. 1a, step2). After a 15-min incubation period, the monolayer samples wererecovered with continuous carbon EM grids (Fig. 1a, step 3),negatively stained and examined in the electron microscope. Theamount of Tf-TfR complex present in the samples increased withthe amount of Ni-NTA lipid in the monolayer (Fig. 1 b–e). Themonolayer containing 2% Ni-NTA lipids (Fig. 1c) showed theoptimum distribution of particles for recording images to be usedfor single-particle averaging techniques. We collected 56 images ofthis sample and selected 9,381 particles that were classified into 100classes. The resulting class averages were consistent with previousstudies of the Tf-TfR complex (9), but as expected from the locationof the His tag protruding from the TfR construct (Fig. 1a), mostaverages showed the ‘‘top view’’ of the complex [supporting infor-mation (SI) Fig. 6a and Fig. 1f, image 1]. Nevertheless, views of thecomplex in other orientations were also observed (SI Fig. 6a andFig. 1f, images 2–6). A plot of the Euler angle distribution con-firmed the preferred orientation of the Tf-TfR complex on themonolayer and the presence of particles in other orientations(Fig. 1g).

After establishing that the Ni-NTA lipids can recruit His-taggedTf-TfR complex to the monolayer surface, we investigated whetherthe interaction would be specific enough to isolate the complexdirectly from cell extracts. We added Tf-TfR complex (ranging from200 ng to 2.5 ng) to OPTI-MEM medium (growth medium for293-T cells) and to mammalian and insect cell extracts. Proteins thatadsorbed to a continuous carbon film and proteins that werespecifically recruited to the Ni-NTA monolayer were visualized by

Author contributions: D.F.K. and T.W. designed research; D.F.K. and D.D. performedresearch; D.F.K. analyzed data; and D.F.K. and T.W. wrote the paper.

The authors declare no conflict of interest.

*To whom correspondence should be addressed. E-mail: twalz@hms.harvard.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0800867105/DC1.

© 2008 by The National Academy of Sciences of the USA

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negative stain EM. In the growth medium, we observed predom-inantly Tf-TfR complexes adsorbing to the carbon film with fewcontaminating proteins (Fig. 2a). In comparison, the solutionsamples of the complex in mammalian (Fig. 2b) and insect cellextracts (Fig. 2c) showed mostly cellular proteins. In contrast, in allcorresponding samples prepared on the Ni-NTA monolayer, wewere able to clearly identify many Tf-TfR complexes, although asignificant background of cellular proteins was also present (Fig. 2d–f). To lower the extent of nonspecific binding, we added variousamounts of imidazole to the aqueous phase of the monolayersamples. To completely suppress nonspecific binding, 10 mMimidazole was required for samples in growth medium (Fig. 2g),whereas mammalian and insect cell extract required 20 mM (Fig.2h) and 50 mM (Fig. 2i) imidazole, respectively. Under theseconditions, the Ni-NTA monolayer could be used to specificallyadsorb as little as 10 ng of the complex from 25-�l samples (0.4�g/ml) of growth medium and cell extracts as concentrated as �7mg/ml. Images of monolayer samples prepared with extracts con-taining �10 ng of the Tf-TfR complex did not show a sufficientnumber of particles (for an incubation time of 15 min) to be usefulfor image processing (data not shown). We subsequently ransamples of the medium and cell extracts containing 10 ng of Tf-TfRcomplex on an SDS/PAGE gel (Fig. 2j, lanes 2–4). The amounts ofTf and TfR were too low to be detected by SimplyBlue stain butproduced faint bands for the His-tagged receptor when a Westernblot was developed with an anti-His antibody (Fig. 2k, lanes 2–4).

To determine the protein composition of the monolayer samplesprepared from the Sf9 extract, we sequentially eluted the boundproteins from 20 monolayer specimens into the same 20-�l drop of300 mM imidazole. The elution was run on an SDS/PAGE gel, andthe entire lane of the gel was excised and analyzed by massspectrometry, which confirmed that the sample contained exclu-sively Tf and TfR (SI Table 1). Although the Tf-TfR complexpurified from insect cell extract by conventional Ni-affinity chro-matography also only contained Tf and TfR when analyzed bySDS/PAGE and mass spectrometry (SI Table 1), it required a much

larger sample volume and a higher concentration of the Tf-TfRcomplex in the extract.

Monolayer-Purified Tf-TfR Complex Is Suitable for Single-ParticleCryo-EM. We tested whether monolayer-purified samples would beamenable to structure determination by single-particle cryo-EM.Samples containing the Sf9 cell extract (7.74 mg/ml) and 10 ng ofthe Tf-TfR complex in the presence of 50 mM imidazole wereoverlaid with a 2% Ni-NTA monolayer. The monolayer sampleswere recovered by using Quantifoil holey carbon grids and quick-frozen in liquid ethane. Despite its molecular mass of only �290kDa, the Tf-TfR complexes were clearly visible in the images butwere sparse (Fig. 3b). We therefore increased the incubation timeto 30 min and increased the amount of Ni-NTA lipids in themonolayer. As we observed previously with negatively stainedspecimens (Fig. 1 b–e), the number of Tf-TfR complexes present inthe samples increased with the amount of Ni-NTA lipid present inthe monolayer. The best distribution of particles in vitrified prep-arations was obtained by using 20% of Ni-NTA lipid in themonolayer (Fig. 3c).

We collected 102 images of frozen-hydrated Tf-TfR monolayerspecimens, selected 19,641 particles and classified them into 100classes. The resulting class averages (SI Fig. 6b and representativeclass averages in Fig. 3e) revealed more variation than those derivedfrom negatively stained specimens. The individual particle imageswere then aligned to a 30-Å reference map of the Tf-TfR complexgenerated from the Protein Data Bank (PDB) coordinates of apreviously determined model [PDB ID code 1SUV (9)]. Afteriterative refinement of the orientation parameters, a plot of theEuler angle distribution revealed that a large fraction of the vitrifiedcomplexes also assumed a preferred orientation (Fig. 3f). However,a sufficient number of complexes adopted different orientations sothat the different views completely defined the structure. The final3D reconstruction had a resolution of 20 Å as determined byFourier shell correlation (SI Fig. 8a), and manual placement of theatomic model into the density map showed that it displayed thesame structural features previously described for the Tf-TfR com-

Fig. 1. Lipid monolayer system for thepreparation of single-particle samples. (a)The His-tagged Tf-TfR complex (Tf in black,TfR in white) in aqueous solution is addedto a 25-�l well in a Teflon block (step 1).Then, 1 �l of a mixture of Ni-NTA and fillerlipids is cast on top of the aqueous solutionto form a monolayer (step 2). After incuba-tion at 4°C, an EM grid is placed on themonolayer sample (step 3). The grid is thenremoved and either negatively stained orfrozen in liquid ethane. (b–e) Negativelystained samples of Tf-TfR complex ad-sorbed to monolayers containing 0% (b),2% (c), 20% (d), and 40% (e) Ni-NTA lipid.(Scale bar: 30 nm.) ( f) Representative classaverages of negatively stained Tf-TfR com-plexes on monolayers. Image 1 shows thepredominant top view of the complex. Theside length of the images is 27 nm. (g)Angular distribution plot showing thatmost of the negatively stained His-taggedcomplexes adopt a preferred orientationon the monolayer.

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plex (Fig. 3g) (9). In addition, a comparison of raw particle imagesand class averages with reprojections from the 3D reconstructiondemonstrated that the density map was consistent with the raw data(SI Fig. 6c).

Application of Monolayer Purification to Ribosomal Complexes. Toassess the use of the monolayer purification method in a real casescenario, we attempted to isolate ribosomal complexes from E. coliextracts. We obtained the hEx1 clone containing an N-terminally

Fig. 2. Monolayer purification of Tf-TfR complex from cell extracts. (a–c) Images of Tf-TfR complex added to 293-T growth medium (a), 293-T cell extract (b),and Sf9 cell extract (c). (d–f ) Corresponding samples after adsorbing the Tf-TfR complex to a lipid monolayer containing 2% Ni-NTA lipid. (g–i) The backgroundin the monolayer samples could be suppressed by adding imidazole to the extracts before casting the monolayer. The imidazole concentrations used were 10mM for growth medium (g), 20 mM for 293-T cell extract (h), and 50 mM for Sf9 cell extract (i). (Scale bar: 30 nm.) ( j and k) The extracts were analyzed by SDS/PAGE( j) and Western blot analysis with anti-His antibody (k). Lane 1, markers; lanes 2 to 4, samples that were imaged in a to c.

Fig. 3. Cryo-EM analysis of monolayer-purified Tf-TfR complex. (a–d) Vitrified samples of Tf-TfR complex added to Sf9 extract containing 50 mM imidazole andadsorbed to monolayers containing 0% (a), 2% (b), 20% (c), and 40% (d) Ni-NTA lipid. (Scale bar: 30 nm.) (e) Representative class averages of vitrified Tf-TfRcomplex on monolayers. The contrast has been inverted for easier comparison with Fig. 1f. The side length of the individual images is 25 nm. ( f) Angulardistribution plot showing that the orientations of the complexes in vitrified ice are more randomly distributed than in negative stain, although a preference forcertain orientations prevails. (g) Different views of the density map of the Tf-TfR complex at 20-Å resolution (gold). The atomic model of the complex [PDB IDcode 1SUV (9)] was placed into the density map (TfR in red, Tf in green). (Scale bar: 2.5 nm.)

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His-tagged construct of the human 60S ribosomal protein L3 (rpl3)from RZPD. Rpl3 is very similar to its E. coli homolog rplC (22%identity) but has 47 additional residues at its N terminus. Wespeculated that this region could potentially act as a spatial linkerbetween the ribosome and the monolayer surface. Based on thestructure of the E. coli ribosome [PDB ID code 1ML5 (10)], rplCis a tightly bound component of the 50S subunit with an accessibleN terminus (Fig. 4a). The rpl3 construct was expressed in E. coli andthe associated ribosomal complexes were purified from the cellextract (Fig. 4b) in parallel by using conventional Ni-affinity chro-matography and Ni-NTA monolayers. Negative stain EM images ofthe rpl3 complexes prepared by using Ni-affinity chromatography(Fig. 4c) showed large particles ranging in size from 20 to 30 nm(consistent with the dimensions of the 50S ribosomal subunit and70S ribosome) as well as a background of smaller particles. Thespecimens prepared by monolayer purification contained similarcomplexes to those obtained with conventional Ni-affinity chro-matography, but less contaminating proteins appeared to bepresent in the background (Fig. 4d). We used SDS/PAGE to furtheranalyze the cell lysate (Fig. 4e, lane 2) and the conventionallypurified (Fig. 4e, lane 3) and monolayer-purified (Fig. 4e, lane 4)rpl3 complexes. The gel was stained with Coomassie blue and rpl3migrating at �48 kDa (expected value of 46.1 kDa) was identifiedby Western blot analysis using an anti-His antibody (Fig. 4f). Massspectrometry analysis was performed on both the monolayer prep-aration and the complexes purified by Ni-affinity chromatography.We found nearly all components of the 70S ribosome to be presentin both samples, although the conventionally purified sample alsocontained several other contaminating proteins, which were notpresent in the monolayer-purified sample (SI Table 2).

We produced frozen-hydrated specimens of the monolayer-purified rpl3 complexes, which were visible in cryo-EM images(Fig. 5a). We collected 591 images of ice-embedded specimensand selected 45,444 particles that were classified into 200 classes(SI Fig. 7). We used FREALIGN (11) to align all class averagesto a 30-Å reference volume of the 50S ribosomal subunit generatedfrom the crystal structure of the 70S ribosome [PDB ID code

1ML5, chains a–x (10)]. The particle images in classes whoseaverages had a cross-correlation coefficient of 0.8 or higher with thereference volume (marked by a white asterisk in SI Fig. 7 andrepresentative class averages shown in Fig. 5b) were combined intoa new dataset. After iterative refinement of the orientation param-eters, these 26,512 particle images produced a 3D reconstruction ofthe 50S ribosomal subunit (Fig. 5c) at a resolution of 22 Å (SI Fig.8b). A plot of the Euler angle distribution revealed that theorientations adopted by the 50S subunits were less biased than thoseadopted by the Tf-TfR complexes (Fig. 5d). Comparison of rawimages and class averages with reprojections from the 3D recon-struction showed that the 3D density map was consistent with theprojection data (Fig. 5e).

DiscussionThe Purification Aspect of Ni-NTA Lipid Monolayers. We establishedthat binding of His-tagged proteins to monolayers depends on thepresence and concentration of the Ni-NTA lipid (Fig. 1) and thatthe monolayer can be used to specifically adsorb complexes fromgrowth medium and cell extracts (Figs. 2 and 4). The addition ofimidazole to the samples before casting the lipid monolayer wasessential to suppress nonspecific binding of proteins to Ni-NTAmonolayers. Although it may be possible to just add a highconcentration of imidazole (e.g., 60 mM) to the extract by defaultbefore casting the Ni-NTA monolayer, high imidazole concentra-tions also will compete with the binding of the His-tagged proteins.It therefore might be best to optimize the required imidazoleconcentration for each specific case. Mass spectrometry analysis ofTf-TfR complex and rpl3 complex showed that samples prepared bymonolayer purification are as clean or cleaner than samples ob-tained by a one-step Ni-affinity purification (SI Tables 1 and 2). Areason for the higher purity of monolayer samples may be the lowerconcentration of Ni-NTA groups in monolayers compared with Niaffinity matrices, rendering it less conducive to nonspecific binding.For monolayer purification to be effective, a 25-�l aliquot of sampleshould contain at least 10 ng of His-tagged protein (0.4 �g/ml),

Fig. 4. Ribosomal complexes with His-tagged rpl3 purified on Ni-NTA monolayers. (a) Space-filling model of the E. coli 50S ribosomal subunit [light blue, PDBID code 1ML5, chains a–x (10)] showing the location of the rplC subunit (green) and its N terminus (red dot and arrowhead). (b) Image of negatively stained E. colicell lysate. (c) Image of negatively stained rpl3-containing complexes purified by Ni-affinity chromatography. (d) Image of negatively stained rpl3-containingcomplexes adsorbed from E. coli cell lysate containing 60 mM imidazole to a 2% Ni-NTA monolayer. (e and f ) SDS/PAGE gel (e) and Western blot detectingHis-tagged rpl3 (f ). Lane 1, markers; lane 2,: E. coli cell lysate; lane 3, rpl3-containing complexes purified by Ni-affinity chromatography; lane 3, rpl3-containingcomplexes eluted from Ni-NTA monolayers. (Scale bar: 30 nm.)

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which should be achievable for most recombinantly expressedproteins.

Single-Particle EM of Lipid Monolayer Samples. For single-particleaveraging techniques, it was essential that a sufficient number ofparticles were being adsorbed to the monolayer while still being wellseparated from each other. The density of bound particles can easilybe adjusted by varying the percentage of Ni-NTA lipids in themonolayer and the incubation time. These parameters will mostlikely need to be adjusted for each specimen. We found it necessaryto use a higher concentration of Ni-NTA lipids and longer adsorp-tion times when producing vitrified specimens. We attribute this tothe shearing off of a portion of the adsorbed proteins from the

monolayer during the plunging of the grid into liquid ethane. Wealso found that particles often were unevenly distributed on themonolayers and clustered in certain areas. EM images of negativelystained samples further revealed that the proteins adsorbed only tomonolayer areas that appeared darker (see, for example, Fig. 1 c–e).It seems that the Ni-NTA lipids, stained darker with uranyl formate,and the filler lipids segregate into separate domains. Different fillerlipids may thus help to obtain more homogeneous particle distri-butions in the future.

Alignment of Tf-TfR images from negatively stained monolayersshowed the preferred orientation of the complex expected from thelocation of the His tag on the N terminus of the stalks of thereceptor. Although vitrified samples still showed some preference

Fig. 5. Cryo-EM analysis of monolayer-purified ribosomal complexes. (a) Vitrified sample of ribosomal complexes containing His-tagged rpl3 from E. coli extractcontaining 60 mM imidazole and adsorbed to a 20% Ni-NTA monolayer. (Scale bar: 30 nm.) (b) Representative class averages of vitrified 50S ribosomal subunitson monolayers. (c) Different views of the density map of the 50S subunit at 22-Å resolution (gray). The atomic model (red) of the 50S subunit [PDB ID code 1ML5,chains a–x (10)] was placed into the density map. (Scale bar: 5 nm.) (d) Angular distribution plot showing that the 50S subunit adopts almost randomly distributedorientations in vitrified ice. (e) Comparison of raw particle images (top row) and class averages (middle row) with reprojections from the 3D reconstruction ofthe 50S subunit (bottom row). The contrast of the raw images and class averages shown in b and e has been inverted, and the side length of the individual imagesis 38 nm.

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in orientation, the particles exhibited enough variation in orienta-tion to calculate a 3D reconstruction. This finding suggests that ifthe His tag is attached to a sufficiently long linker, the complex canadopt almost any orientation, although with different frequency. Italso suggests that the restricted orientations seen in negativelystained specimens are caused by the staining procedure and mostlikely occur upon drying of the grid. Thus, monolayer-purifiedsamples prepared by negative staining are suitable for randomconical tilt 3D reconstruction, whereas vitrified samples are suitablefor common line-based 3D reconstruction approaches. Althoughthe 3D reconstructions we present here have a resolution of �20Å, we believe that the lipid monolayer has no adverse effect on theresolution that can be achieved and that the resolution of thepresented reconstructions could be further improved by addingmore particle images to the datasets. An added advantage of thelipid monolayer is that it renders specimen vitrification very repro-ducible because of the continuous layer of lipids covering the holesin the carbon film. The ice thickness thus remains constant overlarge areas of the grid. The monolayer also introduces only verylittle background noise in the images. The small (�290 kDa) Tf-TfRcomplex is essentially as visible in vitrified monolayer samples(Fig. 3 b–d) as in conventionally vitrified samples (9).

Advantages and Other Potential Applications of Monolayer Purifica-tion. Monolayer purification is rapid and convenient. Besidesavoiding lengthy biochemical purification steps, the short timeperiod between lysing the cells and the final EM specimen shouldbe beneficial for structural analyses of dynamic and unstablecomplexes. The method should also prove useful for structuralstudies on complexes that cannot be expressed in large quantities.

Monolayer purification produces clean samples in terms ofreducing contaminating proteins. However, the Ni-NTA monolayeralso adsorbs every complex that contains the His-tagged protein. Inthe case of His-tagged rpl3, the monolayer not only isolated 50Ssubunits (marked by a white asterisk in SI Fig. 7) but also 70Sribosomes (marked by a white circle in SI Fig. 7) and presumablyvarious smaller complexes that may represent intermediates in theassembly pathway of the ribosome. Monolayer purification willproduce such mixtures of fully and partially assembled complexes inmany cases. When prepared by negative staining, which usuallyresults in preferred orientations of the complexes, the images canbe classified into structurally homogeneous groups and can thusprovide valuable information on stable intermediates in the assem-bly pathway, subcomplexes, and alternative complexes containingthe His-tagged protein. 3D reconstructions of the various speciescan then be calculated by using the random conical tilt approach(12). Structural heterogeneity in EM preparations causes, however,a problem for single-particle reconstructions of vitrified specimen,which may be solved computationally. Alternatively, structuralheterogeneity may also be addressed at the specimen level byHis-tagging specific subunits that are only associated with a par-ticular state of a complex. In this case, the complexes present inmonolayer preparations are certain to be in the state associated

with the tagged subunit. Similarly, by His-tagging either activatorsor substrates, only those complexes that either have the taggedsubstrate or activator bound can be specifically adsorbed to themonolayer. Thus, by carefully choosing the protein to be tagged, itmay be possible to significantly reduce structural heterogeneity inmonolayer preparations even for dynamic biological machines.

High-throughput structure determination of macromolecularcomplexes is a goal of single-particle EM that has not yet beenrealized. As much progress has been made in automation ofelectron microscopic data collection and image processing (13), thelimiting factor for high-throughput EM studies has become thepurification and preparation of complexes suitable for EM studies.The ease and speed with which monolayer samples can be preparedmay help to overcome this limitation. Even if many of the prepa-rations prove to be too heterogeneous for structure determination,the availability of libraries of His-tagged proteins (14) makes itfeasible to produce many samples in a very short time. Themonolayer purification technique thus may remove a substantialhurdle for high-throughput structure determination by single-particle EM.

MethodsPreparation of His-Tagged Tf-TfR and Ribosomal Complexes. Expression ofHis-tagged TfR and rpl3 and preparation of the Tf-TfR complex are described inSI Text.

Conventional Purification of His-Tagged Tf-TfR and Ribosomal Complexes.Purification procedures for His-tagged Tf-TfR and ribosomal complexes byNi-affinity chromatography are described in SI Text.

Preparation of Cell Extracts. Preparation of extracts is described in SI Text.

Adsorption of His-Tagged Proteins to Ni-NTA Lipid-Containing Monolayers. DLPCand 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl-nickel salt (Ni-NTA lipid) were purchased from Avanti Polar Lipids. Eachlipid was reconstituted in chloroform to 1 mg/ml. Teflon blocks containing 18wells, with each well holding a volume of �25 �l, were used to set up monolayersamples. After placing 25 �l of protein solution or cell extract into a well, 1 �l oflipid mixture (DLPC containing the desired percentage of Ni-NTA lipid in chloro-form) was added on top of the aqueous solution to form a monolayer at theair–water interface.Whenindicated,10to60mMimidazole (final concentration)was added to the extracts before they were placed into the wells. The Teflonblocks were incubated in a sealed humid environment at 4°C for 15 min. Mono-layer samples were recovered by placing either a continuous carbon-coated EMgrid or a Quantifoil 2/1 holey grid (Quantifoil Micro Tools) on top of the mono-layer. The grid was gently lifted off with forceps, blotted with Whatman #1 filterpaper (Whatman International) and stained with 0.75% uranyl formate orplunged into liquid ethane. Details on individual monolayer purification exper-iments are provided as SI Text.

Specimen Preparation, EM, and Image Processing. Details are provided in SI Text.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth Grant GM62580 (to Stephen Harrison). The molecular EM facility atHarvard Medical School was established by a generous donation from theGiovanni Armenise–Harvard Center for Structural Biology.

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