凝血酶原 – 因子 II 多克隆抗体 – Affinity PurifiedAntibodies

Affinity 的凝血酶原 – 因子 II 多克隆抗体 – Affinity Purified 是我们凝血酶原 – 因子 II (FII) 抗体家族中最高水平的。在抗原亲和纯化过程中，IgG 已消除任何非特异性免疫球蛋白部分，这增强了剩余免疫球蛋白对靶抗原的特异性。结果是纯度非常高的产品，其滴度明显高于完整或纯化的 IgG。我们的凝血酶原 - 因子 II 多角抗体 - 亲和纯化以含有 50% 甘油 (v/v) 的 HEPES 缓冲盐水溶液形式提供，适用于免疫印迹、细胞免疫染色和多种类型的免疫测定等应用，其中较高信号 -需要使用这种浓缩产品达到信噪比。

宿主动物：绵羊

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仅供研究使用。文件产品插页更多信息安全数据表更多信息相关产品凝血酶原 - 因子 II 配对抗体套装

FII-EIA – 凝血酶原 – 因子 II 配对抗体套装 – 5 板套装

凝血酶原 - 因子 II 多克隆抗体

SAFII-IG – 羊抗人凝血酶原 – 因子 II，纯化 IgG（10.0 毫克小瓶）

凝血酶原 – 因子 II 多克隆抗体 - HRP 缀合

SAFII-HRP – 羊抗人凝血酶原，过氧化物酶缀合 IgG（0.2 毫克小瓶）

缺乏 II 因子的血浆 - 冷冻

FII-DP – 缺乏 II 因子的血浆 – 1.0 mL 和散装

凝血酶原 – 因子 II 多克隆抗体 – HRP 缀合抗体

Affinity 的凝血酶原 – 因子 II 多克隆抗体 – HRP 缀合是从与辣根过氧化物酶缀合的抗血清中纯化的完整 IgG。 IgG-HRP 以含有 50% 甘油 (v/v) 的缓冲稳定剂溶液形式提供。通过分光光度法测定，该缀合物的 Rz 比（Reinheitszahl，A403/A280）≥0.25。总蛋白为 0.2 毫克。

宿主动物：绵羊

应用：该抗体通常用作免疫测定和免疫印迹等应用中的标记一抗。

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FII-DP – II 因子缺陷血浆 – 1.0 mL 和散装

凝血酶原 - II 因子配对抗体套装

FII-EIA – 凝血酶原 – II 因子配对抗体套装 – 5 板套装

凝血酶原 - 因子 II 多克隆抗体

SAFII-IG – 羊抗人凝血酶原 – 因子 II，纯化 IgG（10.0 毫克小瓶）

凝血酶原 - 因子 II 多克隆抗体 -亲和纯化

SAFII-AP – 羊抗人凝血酶原，亲和纯化 IgG（0.5 毫克小瓶）

凝血酶原 – 因子 II 多克隆抗体抗体

Affinity 的凝血酶原 – 因子 II 多克隆抗体是我们凝血酶原 – 因子 II 抗体家族的基础水平。 IgG 的纯度通常为 90%，以含有 50% 甘油 (v/v) 的 HEPES 缓冲盐水溶液形式提供。滴度基本上与起始抗血清相同，并且每个小瓶通常含有从一毫升抗血清中回收的 IgG 量。该凝血酶原 - 因子 II (FII) 多克隆抗体通常用于免疫沉淀、免疫电泳、免疫耗竭和活性中和测定等应用。

宿主动物：羊

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FII-DP – II 因子缺陷血浆 – 1.0 mL 和散装

凝血酶原 - II 因子配对抗体套装

FII-EIA – 凝血酶原 – II 因子配对抗体套装 – 5 块板套装

凝血酶原 - 因子 II 多克隆抗体 - 亲和纯化

SAFII-AP – 羊抗人凝血酶原，亲和纯化 IgG（0.5 毫克小瓶）

凝血酶原 - 因子 II 多克隆抗体- HRP 缀合

SAFII-HRP – 绵羊抗人凝血酶原、过氧化物酶缀合 IgG（0.2 毫克小瓶）

翻译错误：未将对象引用设置到对象的实例。翻译错误：未将对象引用设置到对象的实例。ArticlePDF AvailableThe Affinity Grid: A Pre-fabricated EM Grid for Monolayer PurificationOctober 2008Journal of Molecular Biology 382(2):423-33DOI:10.1016/j.jmb.2008.07.023SourcePubMedAuthors: Deb KellyPennsylvania State University Priyanka D AbeyrathnePriyanka D AbeyrathneThis person is not on ResearchGate, or hasn t claimed this research yet. Danijela DukovskiProteostasis Therapeutics, Inc. Thomas WalzThomas WalzThis person is not on ResearchGate, or hasn t claimed this research yet. Download full-text PDFRead full-textDownload full-text PDFRead full-textDownload citation Copy link Link copied Read full-text Download citation Copy link Link copiedCitations (54)References (14)Figures (4)Abstract and FiguresWe have recently developed monolayer purification as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single-particle electron microscopy (EM) without biochemical purification. Here, we introduce the Affinity Grid, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from Escherichia coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50 S subunit rplC. Ribosomal complexes with or without associated mRNA could be prepared depending on the way the sample was applied to the Affinity Grid . Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50 S ribosomal subunit as well as the 70 S ribosome and 30 S ribosomal subunit from images of the same sample. We established that Affinity Grids are stable for some time in the presence of glycerol and detergents, which allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single-particle EM specimens of soluble complexes and membrane proteins. Affinity Grid and monolayer purification of Tf-TfR complexes from Sf9 cell extract…  Affinity Grid preparation of ribosomal complexes…  3D reconstructions of vitrified ribosomal complexes purified on Affinity Grids…  Affinity Grid purification of His-tagged AQP9… Figures - uploaded by Deb KellyAuthor contentAll figure content in this area was uploaded by Deb KellyContent may be subject to copyright. Discover the world s research20+ million members135+ million publications700k+ research projectsJoin for freePublic Full-text 1Content uploaded by Deb KellyAuthor contentAll content in this area was uploaded by Deb Kelly on Apr 27, 2018 Content may be subject to copyright. The Affinity Grid: A pre-fabricated EM grid for monolayerpurificationDeborah F. Kelly, Priyanka D. Abeyrathne, Danijela Dukovski, and Thomas WalzDepartment of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115,USAAbstractWe have recently developed \"monolayer purification” as a rapid and convenient technique to producespecimens of His-tagged proteins or macromolecular complexes for single particle electronmicroscopy (EM) without prior biochemical purification. Here, we introduce the \"Affinity Grid”, apre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipidsfunctionalized with a Nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored forseveral months under ambient conditions, further simplifies and extends the use of monolayerpurification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomalcomplexes from E. coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli50S subunit rplC. Depending on the way the sample was applied to the Affinity Grid, ribosomalcomplexes with or without associated mRNA could be prepared. Vitrified Affinity Grid specimenscould be used to calculate three-dimensional reconstructions of the 50S ribosomal subunit as well asthe 70S ribosome and 30S ribosomal subunit from images of the same sample. In addition, weestablished that Affinity Grids are stable for some time in the presence of glycerol and detergents.This feature allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilizedmembrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single particleEM specimens of soluble complexes and membrane proteins.Keywordsmonolayer purification; lipid monolayer; affinity purification; single particle; cryo-electronmicroscopyINTRODUCTIONThe biochemical purification of proteins and macromolecular complexes has become the rate-limiting step in structure determination by single particle cryo-electron microscopy (EM). Apurification protocol of a recombinant protein for structural studies typically consists of anaffinity chromatography step followed by size exclusion chromatography. Such a two-steppurification scheme requires an appreciable amount of protein, which cannot always beproduced, and takes several hours, during which time biological complexes can disintegrate.We have recently introduced \"monolayer purification”, a technique that combines proteinpurification with specimen preparation, as a fast and easy way to prepare specimens suitableCorresponding author: T.W. email: twalz@hms.harvard.edu; phone: +1 (617) 432-4090.Publisher s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptJ Mol Biol. Author manuscript; available in PMC 2009 October 3.Published in final edited form as:J Mol Biol. 2008 October 3; 382(2): 423–433. doi:10.1016/j.jmb.2008.07.023.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript for single particle EM, requiring only low expression levels or low concentrations of His-taggedprotein 1. In the monolayer purification technique, the extract of cells expressing a His-taggedprotein, which can be a subunit of a larger assembly, is overlaid with a lipid monolayer thatcontains Ni-NTA lipid, a lipid whose head group is functionalized by Nickel-nitrilotriaceticacid (Ni-NTA). The Ni-NTA lipids recruit the His-tagged proteins to the lipid monolayer,which can then be lifted off with an EM grid covered with a continuous or holey carbon supportfilm and prepared by negative staining or vitrification for subsequent imaging in the electronmicroscope.Here, we introduce the \"Affinity Grid”, a pre-fabricated EM grid with a dried, Ni-NTA lipid-containing monolayer, as a further simplification and extension of the monolayer purificationtechnique. Using His-tagged transferrin-transferrin receptor (Tf-TfR) complex, we show thatthe Affinity Grid produces specimens equivalent to those obtained by monolayer purification.Although Affinity Grids can be incubated with sample for several hours, an incubation timeof only a few minutes usually suffices to produce suitable EM specimens. We also establishthat Affinity Grids are resistant to most detergents for a time that depends on the detergentconcentration, making them useful for isolating membrane proteins. Finally, we use AffinityGrids to isolate ribosomal complexes from an E. coli extract and the water channel AQP9 froma membrane extract of Sf9 insect cells. Since Affinity Grids can be stored under ambientconditions for several months, they can be pre-fabricated and used whenever needed to preparespecimens for single particle EM – within minutes and with minimum effort.RESULTSCharacterization of the Affinity GridWe used His-tagged Tf-TfR complex as a test specimen to characterize the Affinity Grid as atool to prepare specimens for single particle EM. We first wanted to confirm that Affinity Gridsproduce samples of the same quality as those prepared with our recently introduced monolayerpurification technique 1. We added His-tagged Tf-TfR complex to Sf9 cell extract (6 mg/mlprotein, 60 mM imidazole) to a final concentration of 0.15 μg/ml. 3 μl of this mixture wasadded to a grid covered with a holey carbon film and a dried lipid monolayer containing 20%Ni-NTA lipid, which we refer to as a \"20% HC Affinity Grid” (a grid covered with a continuouscarbon film and a lipid monolayer containing 2% Ni-NTA lipid would be a \"2% CC AffinityGrid”). After a 2-minute incubation, the grid was blotted and vitrified. Images of the AffinityGrid sample (Fig. 1b) looked virtually identical to images taken of Tf-TfR complexes preparedby monolayer purification (Fig. 1a). However, while the Tf-TfR complexes tended to clusterwhen prepared by monolayer purification (already reported in 1), they were more evenlydistributed on the Affinity Grid. Most of the particles attached to the Affinity Grid showed thecharacteristic shape of the Tf-TfR complex, suggesting the absence of contaminating proteins.The number of particles present on the Affinity Grid can be controlled by adjusting thepercentage of Ni-NTA lipid in the lipid mixture used to prepare the Affinity Grid or, moreeasily, by changing the time the Affinity Grid is incubated with the saple solution. Furthermore,no proteins bound to Affinity Grids when Ni-NTA lipids were omitted from the monolayer(data not shown), consistent with previous results obtained with the monolayer purificationtechnique 1. To confirm the purity of Affinity Grid samples, 20 samples on 20% CC AffinityGrids were eluted into the same 20-μl drop of 300 mM imidazole. For comparison, we alsoeluted 20 monolayer-purified samples. SDS-PAGE analysis showed a smear over the entirelane for the insect cell extract used as input (Fig. 1c, lane 1), while the samples eluted from themonolayer purification grids (Fig. 1c, lane 2) and the Affinity Grids (Fig. 1c, lane 3) showedonly two bands corresponding to Tf and TfR, which have a very similar molecular weight of~75 kDa. A Western blot of the same samples developed with an anti-His antibody (Fig. 1d)showed two bands for the His-tagged TfR, one at ~75 kDa and one in the higher molecularKelly et al. Page 2J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript weight range, presumably representing dimeric TfR. The entire lanes 2 and 3 were excisedfrom the gel shown in Figure 1c and analyzed by mass spectrometry as previously described1. The results confirmed that only Tf and TfR were present in the samples eluted from gridsprepared by monolayer purification and from Affinity Grids (Supplementary Table 1). AffinityGrids reproducibly gave the same results, even if Affinity Grids were used that were stored forup to 6 months under ambient conditions.To test the potential use of Affinity Grids for preparing membrane protein samples, we testedtheir tolerance for detergents. We prepared His-tagged Tf-TfR complex (0.15 μg/ml) in bufferscontaining either 1% OG, 0.2% DM, 0.02% DDM, 0.03% Triton X-100, 0.014% Tween 20,0.5% CHAPS, 0.2% Fos-choline 11 or 0.1% digitonin and applied 3-μl aliquots of thesesamples to 2% CC Affinity Grids. Samples were incubated for 30 seconds, 1, 2, 5, 10, 20, 30and 60 minutes prior to negative staining and assessed by visual inspection in the electronmicroscope. We found that Affinity Grids were stable in the presence of 1% OG, 0.2% DMand 0.02% DDM for up to 30 minutes, producing useful specimens for single particle EM.After 60 minutes of incubation, the lipid monolayer started to show signs of degradation, suchas the appearance of small holes in the lipid film or small (~5 – 10 nm), round lipid vesicles,which are easily distinguished from protein due to their very low contrast. This resulted inspecimens with little or no Tf-TfR complexes present on the grid. In the presence of 0.03%Triton X-100 and 0.2% Fos-choline 11, the lipid monolayer was stable for up to 10 minutesbefore it began to dissolve. Samples in 0.5% CHAPS could only be incubated for 2 minutesand samples in 0.014% Tween-20 for 30 seconds before the monolayer degraded. 0.1%digitonin proved incompatible with Affinity Grids, even at incubation times as short as 30seconds. To determine whether the detergent concentration has an influence on the stability ofthe Affinity Grid, we prepared Tf-TfR complex in 1, 2, 3, 4 and 5% OG on 2% CC AffinityGrids and incubated them for the same times prior to negative staining. We found that AffinityGrids can tolerate up to 2% OG for 20 – 30 minutes before substantial degradation of the lipidmonolayer occurred, while 3% OG already began to destabilize the monolayer after 2 minutes.OG concentrations of 4 – 5% were incompatible with Affinity Grids even for very shortincubation times. The results of these experiments (summarized in Table 1) suggest thatAffinity Grids are compatible with most detergents, at least for some time at detergentconcentrations not too far above the critical micelle concentration.We also tested the compatibility of Affinity Grids with glycerol, as glycerol is often used tostabilize protein complexes and is employed in certain cryo-negative staining protocols. Weprepared His-tagged Tf-TfR complex (0.15 μg/ml) in buffer solution containing 1, 2, 3, 4 and5% glycerol and applied 3-μl aliquots of each sample to 2% CC Affinity Grids. Samples wereincubated for 1, 5, 10 and 15 minutes prior to extensive washing and negative staining. AffinityGrids were stable in the presence of up to 5% glycerol for at least a 5-minute incubation period.After 10 minutes of incubation, only glycerol concentrations of less than or equal to 3%produced useful samples with only minor lipid layer degradation. By 15 minutes, all glycerolconcentrations tested caused substantial degradation of the lipid monolayer (see Table 2 for asummary of these results).Affinity Grid purification of ribosomal complexesWe previously demonstrated that native 50S ribosomal subunits containing His-tagged humanrpl3 can be purified from E. coli extracts using monolayer purification 1. We now tested theAffinity Grid with the same system. We added a 3-μl drop of E. coli extract (~3 mg/ml) to a2% CC Affinity Grid (Fig. 2e). After an incubation of 2 minutes, the sample was negativelystained and imaged in the electron microscope (Fig. 2a). The images showed the same kind ofcomplexes, 20 to 30 nm in size, that we previously observed with specimens produced byconventional Ni-affinity chromatography and monolayer purification 1. To assess theKelly et al. Page 3J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript composition of the complexes seen in the images, protein was eluted from 20 samples on 20%CC Affinity Grids and analyzed by mass spectrometry. The same proteins were present aspreviously identified in monolayer-purified samples 1; all of them were known ribosomalsubunits and no contaminating proteins could be identified (Supplementary Table 2).To produce vitrified specimens, we added a 3-μl drop of E. coli extract to a 20% HC AffinityGrid. After a 2-minute incubation, the sample was blotted, quick-frozen in liquid ethane andimaged in the electron microscope (Fig. 2b). A comparison with images of vitrified samplesprepared by monolayer purification (Fig. 2c) revealed distinct differences. First, as previouslyobserved, clustering of the particles often seen in samples prepared by monolayer purificationdid not occur on the Affinity Grid. Second, while both samples contained mostly particlesconsistent in size with the 50S ribosomal subunit or the 70S ribosome, the Affinity Gridspecimen also contained smaller particles that would be consistent in size and shape with a30S ribosomal subunit (black arrows in Fig. 2b). Third, and most notably, the images obtainedwith the Affinity Grid sample showed that many of the ribosomal complexes were attached to~15 Å thick, string-like densities, which we thought most likely represented mRNA strands.To test this interpretation, we incubated the extract with RNAse A for 30 minutes prior toapplying it to an Affinity Grid. The images from this preparation no longer showed the string-like densities associated with the ribosomal complexes (Fig. 2d), confirming that theyrepresented mRNA. mRNA was consistently present in Affinity Grid preparations of ribosomalcomplexes (Fig. 2b), but was always missing from preparations produced by monolayerpurification (Fig. 2c). To obtain an Affinity Grid sample in a manner more resembling the waya monolayer purification sample is prepared, we placed 25 μl of extract into a well in a Teflonblock and placed a 20% HC Affinity Grid on top of the extract (as illustrated in Fig. 2f). Aftera 30-minute incubation, the Affinity Grid was lifted off, blotted and vitrified. Images of thispreparation did not show mRNA, while mRNA was visible on an Affinity Grid specimenprepared by pippetting a drop of the same extract onto an Affinity Grid (as illustrated in Fig.2e) at the same time (data not shown). These results indicate that the presence of mRNA inAffinity Grid preparations depends on the exact way the sample was applied to the AffinityGrid.To calculate structures of ribosomal complexes, we selected 52,507 particles from 274 imagesof vitrified Affinity Grid specimens containing mRNA and classified them into 200 classes(Supplementary Fig. 1). To account for the heterogeneity in the class averages due to thepresence of different ribosomal complexes, we created 30-Å density maps of the 30S, 50S and70S ribosomal complexes based on the crystal structure of the E. coli 70S ribosome (pdb code:1ML5; 2). We used these reference volumes to sort the class averages into groups representingthe 50S ribosomal subunit (representative class averages shown in Fig. 3a), the 30S ribosomalsubunit (representative class averages shown in Fig. 3e) and the 70S ribosome (representativeclass averages shown in Fig. 3i) using the normalized cross-correlation routine implementedin SPIDER 3. We then used FREALIGN 4 to calculate 3D reconstructions of the 50S subunit(Fig. 3d; 23,680 particles in the final map) at a resolution of 21 Å (Fig. 3c), the 30S subunit(Fig. 3h; 7,226 particles in the final map) at a resolution of 24 Å (Fig. 3g), and the 70S ribosome(Fig. 3l; 3,178 particles in the final map) at a resolution of 28 Å (Fig. 3k). The Euler angledistribution for each reconstruction showed that the particle orientations sampled the entire 3Dspace (Figs. 3b, f and j). Manual placement of the atomic models for each ribosomal complexinto the corresponding density map demonstrated that the structural features of the densitymaps were consistent with the crystal structures (Fig. 3d, h and l).Affinity Grid purification of AQP9Since the Affinity Grid proved to be sufficiently resistant to OG, we tested whether we coulduse it to isolate AQP9 from a membrane extract of Sf9 insect cell. Sf9 cells over-expressingKelly et al. Page 4J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript His-tagged AQP9 were lysed and the membrane fraction was solubilized with 2% OG.Imidazole was added to a final concentration of 60 mM and the detergent-solubilizedmembranes were applied to a 2% CC Affinity Grid. In parallel, a conventional purification wasperformed for the same membrane fraction using Ni-affinity and gel filtration chromatography.Negatively stained specimens were prepared for the two samples and examined in the electronmicroscope.Negatively stained specimens of the Sf9 membrane extract prepared in the conventional wayshowed a variety of proteins, making it impossible to identify individual AQP9 tetramers (Fig.4a). In contrast, specimens prepared by conventional Ni-affinity and gel filtrationchromatography (Fig. 4b) and using an Affinity Grid (Fig. 4c) showed largely homogeneousparticle populations. To assess the purity of the sample produced with the Affinity Grid, theprotein was eluted from 20 samples on 40% CC Affinity Grids and analyzed by SDS-PAGE(Fig. 4d, lane 2) and Western blotting (Fig. 4e, lane 2) as well as by mass spectrometry. Forcomparison, AQP9 purified by conventional Ni-affinity chromatography was subjected to thesame analyses (lane 1 in Figs. 4d and 4e). The SDS-PAGE gel and Western blot of the twosamples show the two bands representing unglycosylated (~32 kDa) and glycosylated AQP9(~35 kDa) that have been previously observed 5 as well as dimeric AQP9 (~68 kDa). Inaddition, the mass spectrometry results confirmed that AQP9 was the only protein present inthe two samples (Supplementary Table 3). We then collected 15 images each of thechromatographically and Affinity Grid purified samples and selected 9,526 and 10,292particles, respectively, that were each classified into 50 classes. The class averages of OG-solubilized AQP9 obtained with the Affinity Grid specimen (Fig. 4g) appeared to be of thesame quality as those obtained with conventionally prepared sample (Fig. 4f). In both caseswe observed particles about ~10 nm in size with a central stain accumulation, which lookedlike those previously reported for single particles of AQP9 5. Because of the small size of theAQP9 tetramer (~130 kDa), we did not attempt to prepare vitrified specimens of AQP9, butwe are optimistic that it should be possible, with sufficiently large membrane proteins, toprepare vitrified specimens using Affinity Grids.DISCUSSIONAffinity Grids in comparison to monolayer purificationWe have recently introduced monolayer purification as an easy and rapid technique to purifya His-tagged protein or macromolecular complex from cell extract while simultaneouslypreparing a specimen suitable for single particle EM 1. While it is easy to cast a monolayerover a cell extract and to then pick it up with an EM grid, it does require this procedure beperformed for every sample, potentially creating a threshold for the use of monolayerpurification. We therefore set out to test whether we could produce an Affinity Grid featuringa dried, pre-deposited monolayer containing Ni-NTA lipids on a carbon-coated EM grid. Ifsuccessful, the Affinity Grid would make it possible to simply apply a drop of extract to thepre-fabricated grid, which would specifically adsorb only His-tagged proteins while otherproteins would be removed during the washing of the grid. Our test with His-tagged Tf-TfRand ribosomal complexes showed that the Affinity Grid indeed worked and produced samplesthat were of the same purity as those produced with monolayer purification. However,preparing an Affinity Grid sample takes much less time and is as convenient as preparing anormal EM grid while not requiring any plasma cleaning or glow discharging of the grid priorto sample application. Particularly useful is the fact that Affinity Grids are stable and can bestored under ambient conditions for at least six months. Thus, Affinity Grids can be producedat any time and used when needed.Kelly et al. Page 5J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Affinity Grids extend the applicability of monolayer purificationMonolayer purification depends on the formation of a lipid monolayer at the air-water interface.Since detergents and glycerol generally dissolve lipid monolayers (personal experience), thesesubstances are incompatible with monolayer purification. Detergents are, however, used in thestandard protocols to prepare yeast extracts and they are occasionally added in lowconcentrations to protein solutions to minimize aggregation through hydrophobic interactions.Detergents are also used to solubilize membrane proteins from cell membranes. Glycerol isanother common additive that stabilizes proteins and complexes in solution, and it is alsoemployed in certain cryo-negative staining procedures 6. The incompatibility of lipidmonolayers with detergents and glycerol thus limits the use of monolayer purification. Sincethe lipid monolayer on the Affinity Grid is adsorbed to a carbon film, we propose that thisinteraction may be the reason why the lipid monolayer is more resistant to detergents andglycerol. With the exception of digitonin, which is known to be particularly potent insolubilizing lipids 7 and destroys the Affinity Grid almost immediately, the Affinity Grid isstable in most detergents for at least some period of time. The time of stability increases withdecreasing detergent concentration. Similarly, Affinity Grids are only useful for a limited timeif the sample contains glycerol, with the lifetime of the Affinity Grid again increasing withdecreasing glycerol concentrations. As Affinity Grids were sufficiently stable in the presenceof 2% OG, they could be used to directly adsorb His-tagged AQP9 from an insect cellmembrane extract. For samples containing more aggressive detergents and/or a highconcentration of glycerol, the best strategy would presumably be to minimize the requiredincubation time by using an Affinity Grid with a high percentage of Ni-NTA lipid. Analternative solution to this problem would be to prepare Affinity Grids with a lipid monolayerthat contains fluorinated lipids as the filler lipid. Fluorinated lipids are more resistant todetergents 8 and have already been used to form two-dimensional (2D) crystals on lipidmonolayers 9. Although not yet tested, fluorinated lipids, which are not commercially availableat this time, may also be more resistant to glycerol.When monolayer purification is used to prepare a specimen, the particles adsorbed to the lipidmonolayer often seem to have a tendency to cluster (Figs. 1a and 2c). Interestingly, suchclustering was not observed with samples prepared on Affinity Grids (Figs. 1b and 2b). Lipidmonolayers are fluid and proteins adsorbing to them can easily diffuse, a feature that isexploited in the 2D crystallization of proteins on lipid monolayers 10. By contrast, the lipidsconstituting the monolayer on the Affinity Grid are attached to the carbon film. We hypothesizethat this attachment makes the monolayer not only more resistant to detergents and glycerol,but also prevents or at least substantially reduces diffusion of the lipids. Clustering of proteinson monolayers thus appears to be due to affinity of the proteins for each other, with the lipidsenabling the proteins to find each other by diffusion. Since the lipids in the monolayer areattached to the carbon film of the Affinity Grid, they are presumably prevented from diffusingthus also preventing particle clustering.In summary, the Affinity Grid makes monolayer purification compatible for use withmembrane proteins and protein solutions containing glycerol, and it is superior to monolayerpurification in most cases because it prevents particle clustering. Once the technique forproducing Affinity Grids has been mastered, the preparation of the grids is highly reproducible,and the grids are usually almost completely covered with the lipid monolayer.Affinity Grid samples of ribosomal complexes differ depending on the preparation methodUnexpectedly, Affinity Grid samples of ribosomal complexes looked quite different fromsamples prepared by monolayer purification. Most notably, the ribosomal complexes were stillassociated with mRNA. The possibility that the RNA was simply degraded in the extract usedto prepare the sample by monolayer purification was ruled out by preparing monolayer andKelly et al. Page 6J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Affinity Grid samples from the same extract. mRNA was again present in the Affinity Gridsample but not in the monolayer purification sample. Since preparing a monolayer purificationsample takes about 20 minutes while preparing an Affinity Grid requires only about 2 minutes,another possibility was that the RNA was degraded in the additional time it took to prepare thegrid by monolayer purification. However, when the Affinity Grid sample was prepared oncethe preparation of the monolayer purification grid was completed, the outcome did not change.Finally, we prepared an Affinity Grid sample by placing it onto the top of cell extract (Fig. 2f)rather than to place a drop of extract onto the Affinity Grid (Fig. 2e). In this preparation, mRNAwas no longer visible. The reason why the two different procedures produce different samplesis currently not clear, but the results were reproducible. It may be that placing extract onto theAffinity Grid is more gentle and thus allows the mRNA to remain on the monolayer, whilelifting the Affinity Grid from the surface of the extract may be harsher and rip the mRNA offthe monolayer. An alternative explanation may be that the surface tension at the air-waterinterface disrupts the RNA strands present in the sample when the specimen is prepared byplacing the Affinity grid on top of the sample and then lifting it off from the aqueous solution.Affinity Grid samples produced by placing cell extract on the grid showed not only the presenceof mRNA, in addition to 50S and 70S complexes, but also revealed 30S ribosomal complexes.Since the 30S complex does not contain a His-tagged subunit (and accordingly was absent insamples prepared by monolayer purification 1), the only possibility for it to be present inAffinity Grid samples is that they were bound to mRNAs that, in turn, were adsorbed to thelipid monolayer through His-tagged ribosomal complexes. The gentleness of the Affinity Gridpreparation thus seems to allow the preparation of large, functional, macromolecularassemblies that usually disintegrate during specimen preparation. The Affinity Grid may thusopen a new avenue to the visualization of complex biological assemblies by cryo-EM orelectron tomography that were not possible to isolate with previously available techniques.MATERIALS AND METHODSExpression of rpl3, AQP9, TfR and production of Tf-TfR complexRpl3 was expressed in E. coli, TfR was expressed in 293-T cells, and the Tf-TfR complex wasproduced as described in 1. AQP9 was expressed in Sf9 cells as described in 5.Preparation of cell and membrane extractsSf9 and E. coli cell extracts were prepared as described in 1. Membrane extract from Sf9 cellswas produced by centrifugation of 50 ml Sf9 cell extract at 100,000g for 30 minutes at 4°C.The pellet containing the cell membranes was homogenized in 100 ml buffer (20 mM Tris, pH8.0, 300 mM NaCl) containing 2% octyl-β,D-glucoside (OG) (Anatrace, Inc., Maumee, OH).The homogenate was centrifuged at 100,000g for 30 minutes at 4°C, and the supernatant wasused for Affinity Grid experiments.Conventional purification of His-tagged Tf-TfR complexConventional Ni-affinity purification of His-tagged Tf-TfR complex was performed asdescribed in 1.Preparation of Affinity Grids1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) and 1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl)iminodi acetic acid] succinyl-nickel salt (Ni-NTA lipid) werepurchased from Avanti Polar lipids (Alabaster, AL). Each lipid was reconstituted in chloroformto 1 mg/ml. A 25-μl buffer aliquot (20 mM Hepes, pH 7.9, 150 mM NaCl) was placed into thewell of a Teflon block, and 1 μl of a lipid mixture (DLPC containing the desired percentageKelly et al. Page 7J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript of Ni-NTA lipid in chloroform) was added on top of the aqueous solution to form a lipidmonolayer at the air-water interface. The Teflon block was incubated in a sealed humidenvironment at 4°C for 15 minutes. A copper EM grid (400 mesh, Ted Pella, Redding, CA)covered with a continuous carbon film or a Quantifoil 2/1 holey carbon grid (400 mesh,Quantifoil Micro Tools GmbH, Germany) was placed onto the lipid monolayer. The grid wasgently lifted off with forceps, blotted from the side (perpendicular to the grid) with Whatman#1 filter paper (Whatman International Ltd, Middlesex, England) and allowed to air-dry. In thecase of Quantifoil 2/1 holey carbon grids, a thin layer of carbon was evaporated onto the carbonside of the grid for stabilization during storage. Affinity Grids prepared in this way were storedfor up to 6 months in a closed grid box (Ted Pella) under ambient conditions of roomtemperature (~25°C) and humidity (not controlled) before use. To specify the type of AffinityGrids used for each set of experiments, we adapt the following nomenclature: X% CC/HCAffinity Grid, where the percentage denotes the proportion of Ni-NTA lipid in the monolayerand CC or HC denotes continuous or holey carbon film.Affinity Grid purification of His-tagged Tf-TfR complex, ribosomal complexes and AQP9Tf-TfR complex—2 μl of Tf-TfR complex (0.03 mg/ml) in 20 mM Hepes, pH 7.4, 150 mMNaCl was added to 38 μl of Sf9 cell extract (6 mg/ml). This mixture was diluted 1:10 usingthe same buffer, and a 3 μl aliquot was applied to a 20% HC Affinity Grid. The sample wasincubated on the grid for 2 minutes prior to blotting and vitrification. Likewise, 2 μl of Tf-TfRcomplex was added to 38 μl of buffer solution (20 mM Hepes, pH 7.4, 150 mM NaCl)containing either glycerol (1 – 5% final concentration) or one of the following detergents: 1 –5% OG (Anatrace), 0.2% n-decyl-β,D-maltoside (DM) (Anatrace), 0.02% n-dodecyl-β,D-maltoside (DDM) (Anatrace), 0.03% Triton X-100 (EMD Bioscsciences, San Diego, CA),0.014% Tween 20 (EMD Biosciences), 0.5% CHAPS (Anatrace), 0.2% Fos-choline 11(Anatrace) and 0.1% digitonin (Sigma-Aldrich, St. Louis, MO). 3 μl of a 1:10 dilution of Tf-TfR complex in solutions containing these detergents or glycerol was added to a 2% CCAffinity Grid and incubated for various times prior to negative staining.Ribosomal complexes—Based on previous results 1, 60 mM imidazole and 20 mMMgCl2 (final concentrations) were added to 1 ml of E. coli extract containing His-tagged rpl3.3 μl of this mixture was placed on a 2% CC Affinity Grid (for negative staining) or a 20% HCAffinity Grid (for vitrification). Samples were incubated for 2 minutes, blotted from the sideand either negatively stained or vitrified.In addition, 25-μl aliquots of the same mixture were placed into two tubes, and 1 μl of RNAseA (Ambion, Inc., Austin, TX) (~ 0.5 units) was added to one tube while 1 μl of buffer wasadded to the other tube. The samples were incubated for 30 minutes at 4ºC. Two 10-μl aliquotsfrom each tube were placed into wells of a teflon block. One of the two wells from each samplewas overlayed with a 20% Ni-NTA lipid monolayer while 20% HC Affinity Grids were placedon top of the other two wells and later recovered. In parallel, 3 μl aliquots of the mixtures withand without RNAse A were added to two 20% HC Affinity Grids. All samples were incubatedfor 30 minutes. For the samples in the teflon block, monolayers were recovered using Quantifoilholey carbon grids while the Affinity Grids were simply lifted off the wells. All samples werevitrified.AQP9—60 mM imidazole (final concentration) was added to 1 ml solubilized Sf9 membranes.3 μl of this mixture was placed on a 2% CC Affinity Grid and incubated for 5 minutes. Thegrid was washed with 7 drops of MilliQ water prior to negative staining.Kelly et al. Page 8J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Sample elution from Affinity GridsA sample was prepared on a 20% CC Affinity Grid, excess solution was blotted off with filterpaper and the grid was then incubated for 2 minutes with 20 μl of 300 mM imidazole. The 20-μl drop was recovered using a pipetteman and added to the next Affinity Grid sample. In thisway, proteins adsorbed to 20 Affinity Grids were eluted into the same 20-μl volume of 300mM imidazole.SDS-PAGE, Western blotting and mass spectrometrySDS-PAGE—Samples were run on 10% SDS-PAGE gels and stained either with Coomassieblue or SimplyBlue stain (Invitrogen Corporation, Carlsbad, CA). Western blotting: His-taggedproteins were detected with anti-His antibody (GE Healthcare, Buckinghamshire, UK) anddeveloped by the alkaline phosphatase method using the Sigma Fast system (Sigma-Aldrich,St. Louis, MO).Protein assay— Protein concentrations were determined using the BCA protein assay kit(Pierce, Rockford, IL).Mass spectrometry—Entire lanes were excised from SimplyBlue stained SDS-PAGE gelsand analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) in the TaplinBiological Mass Spectrometry Facility at Harvard Medical School.Specimen preparationNegative staining—For conventionally purified protein samples, grids were negativelystained with 0.75% uranyl formate as described in 6 and grids of monolayer-purified sampleswere stained as described in 1. For Affinity Grids, 3-μl sample aliquots were placed on theAffinity Grids and incubated for various times. The grids were blotted from the side, washedwith one drop of 0.75% uranyl formate and stained for 20 seconds with another drop of 0.75%uranyl formate. Affinity Grids of specimens in detergent solution were washed with 7 dropsof MilliQ water before staining and grids of specimens in glycerol solution with 15 drops ofMilliQ water.Vitrification—Grids of monolayer-purified samples were vitrified as described in 1. ForAffinity Grids, 3-μl sample aliquots were placed on Affinity Grids, which were blotted for 3seconds and plunged into liquid ethane using a Vitrobot (FEI Company, Hillsboro, Oregon)operating at 22°C and 65% relative humidity.Electron microscopyNegatively stained specimens were imaged in an FEI Tecnai 12 electron microscope (FEI,Hillsboro, OR) equipped with a LaB6 filament and operated at an acceleration voltage of 120kV. Images were recorded on imaging plates under low-dose conditions at a nominalmagnification of 67,000x and a defocus value of about −1.5 μm. Imaging plates were scannedwith a Ditabis scanner (Pforzheim, Germany) using a step size of 15 μm, a gain setting of20,000 and a laser power setting of 30%. The images were binned over 2 x 2 pixels for a finalsampling of 4.5 Å/pixel at the specimen level.Grids of vitrified specimens were transferred into an FEI F20 electron microscope equippedwith a field emission gun using an Oxford cryo-specimen holder, maintaining a temperatureof −180°C. Samples were examined at an acceleration voltage of 200 kV and images wererecorded on Kodak SO-163 film at a nominal magnification of 50,000x using low-doseprocedures and a defocus ranging from −2 to −4 μm. Film negatives were developed for 12minutes with full-strength Kodak D-19 developer at 20°C. Micrographs were digitized with aKelly et al. Page 9J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Zeiss SCAI scanner (Carl Zeiss Inc., Oberkochen, Germany) using a step size of 7 μm andbinned over 3 x 3 pixels for a final sampling of 4.2 Å/pixel at the specimen level.Image processingRibosomal complexes—WEB, the display program associated with the SPIDER softwarepackage 3, was used to select 52,507 particles from 274 images of vitrified specimens ofribosomal complexes, in which mRNA could also be seen. The particles were windowed intoindividual images of 90 x 90 pixels. Using the SPIDER software package, the particles werelow-pass filtered to 20 Å, rotationally and translationally aligned and subjected to 10 cycles ofmulti-reference alignment. Each round of multi-reference alignment was followed by K-meansclassification into 200 classes. The references used for the first multi-reference alignment wererandomly chosen from the raw images.The EMAN software package 11 was used to calculate reference volumes filtered to 30 Åresolution based on the atomic model of the 70S ribosome (pdb code: 1ML5) 2 for the 70Sribosome (all chains) and the 50S (chains a - x) and 30S (chains A and C - X) ribosomalsubunits. Re-projections from the reference volumes were calculated at 4° intervals and cross-correlated with the 200 experimental class averages. Class averages were assigned either tothe 70S, 50S or 30S ribosomal complexes depending on a normalized cross-correlationcoefficient of 0.8 or higher with re-projections of the respective reference volume. The particlesbelonging to the class averages were then used to create 3 image stacks corresponding to the70S (3,973 particles from 10 classes), 50S (29,600 particles from 81 classes) or 30S (9,032particles from 24 classes) ribosomal complexes.Three independent reconstructions were calculated with FREALIGN version 7.05 4, whichwas used to determine and refine the orientation parameters for each particle and to correct forthe contrast transfer function (CTF) of the microscope. The correct defocus value for eachparticle image was deduced from the position of each particle in the image and the tilt anglesand defocus values of the images, which were determined with CTFTILT 12. FREALIGN wasfirst run for one round using mode 3 (systematic parameter search) with an angular step of 7°to determine initial orientation parameters for each particle relative to the reference model. Theresulting parameters were iteratively refined over 15 additional cycles running in mode 1 (localparameter refinement) including data in the 200 − 10 Å resolution range. Only particles witha weighted cross-correlation coefficient better than 0.8 were included in the finalreconstructions, which were 23,680 particles for the 50S subunit, 7,226 particles for the 30Ssubunit, and 3,178 particles for the 70S ribosome. The final density maps were low-pass filteredaccording to their respective resolutions, which were estimated by Fourier shell correlation(FSC) with the FSC = 0.5 cut-off criterion 13.AQP9—9,526 particles from the conventionally purified sample and 10,292 particles fromthe Affinity Grid sample were selected from 15 images of each specimen and windowed into64 x 64 pixel images. The particles in each data set were classified into 50 classes as describedabove.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgementsThis work was supported by National Institutes of Health grant GM62580 (to S. C. Harrison). The molecular EMfacility at Harvard Medical School was established with a generous donation from the Giovanni Armenise HarvardCenter for Structural Biology. We thank Matthias Wolf for discussions and advice on image processing.Kelly et al. Page 10J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript References1. Kelly DF, Dukovski D, Walz T. Monolayer purification: a rapid method for isolating protein complexesfor single-particle electron microscopy. Proc Natl Acad Sci U S A 2008;105:4703–4708. [PubMed:18347330]2. Klaholz BP, Pape T, Zavialov AV, Myasnikov AG, Orlova EV, Vestergaard B, Ehrenberg M, van HeelM. Structure of the Escherichia coli ribosomal termination complex with release factor 2. Nature2003;421:90–94. [PubMed: 12511961]3. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A. SPIDER and WEB: processingand visualization of images in 3D electron microscopy and related fields. J Struct Biol 1996;116:190–9. [PubMed: 8742743]4. Grigorieff N. FREALIGN: high-resolution refinement of single particle structures. J Struct Biol2007;157:117–25. [PubMed: 16828314]5. Viadiu H, Gonen T, Walz T. Projection map of aquaporin-9 at 7 Å resolution. J Mol Biol 2007;367:80–88. [PubMed: 17239399]6. Ohi M, Li Y, Cheng Y, Walz T. Negative staining and image classification - powerful tools in modernelectron microscopy. Biol Proced Online 2004;6:23–34. [PubMed: 15103397]7. Moore RJ, Wilson JD. Extraction of the reduced nicotinamide adenine dinucleotide phosphate:delta4-3-ketosteroid-5-α-oxidoreductase of rat prostate with digitonin and potassium chloride.Biochemistry 1974;13:450–456. [PubMed: 4810062]8. Lebeau L, Lach F, Venien-Bryan C, Renault A, Dietrich J, Jahn T, Palmgren MG, Kühlbrandt W,Mioskowski C. Two-dimensional crystallization of a membrane protein on a detergent-resistant lipidmonolayer. J Mol Biol 2001;308:639–647. [PubMed: 11350166]9. Levy D, Chami M, Rigaud JL. Two-dimensional crystallization of membrane proteins: the lipid layerstrategy. FEBS Lett 2001;504:187–193. [PubMed: 11532452]10. Uzgiris EE, Kornberg RD. Two-dimensional crystallization technique for imaging macromolecules,with application to antigen-antibody-complement complexes. Nature 1983;301:125–129. [PubMed:6823289]11. Ludtke SJ, Baldwin PR, Chiu W. EMAN: semiautomated software for high-resolution single-particlereconstructions. J Struct Biol 1999;128:82–97. [PubMed: 10600563]12. Mindell JA, Grigorieff N. Accurate determination of local defocus and specimen tilt in electronmicroscopy. J Struct Biol 2003;142:334–347. [PubMed: 12781660]13. Bottcher B, Wynne SA, Crowther RA. Determination of the fold of the core protein of hepatitis Bvirus by electron cryomicroscopy. Nature 1997;386:88–91. [PubMed: 9052786]Kelly et al. Page 11J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 1. Affinity Grid and monolayer purification of Tf-TfR complexes from Sf9 cell extract(a, b) Images of Tf-TfR complex added to Sf9 cell extract containing 60 mM imidazole andprepared by monolayer purification using a 20% Ni-NTA lipid monolayer (a) or a 20% HCAffinity Grid (b). The images of the vitrified samples show that the Tf-TfR complexes aremore evenly distributed on the Affinity Grid. Scale bar is 30 nm. (c) SimplyBlue stained SDS-PAGE gel. Lane 1: cell extract containing Tf-TfR complex; lane 2: protein eluted from 20monolayer purification samples; lane 3: protein eluted from 20 Affinity Grid samples. (d)Western blot analysis of the gel shown in (c) developed with anti-His antibody to detect His-tagged TfR. The band at ~75 kDa represents monomeric TfR, whereas the band at ~150 kDamost likely represents dimeric TfR.Kelly et al. Page 12J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 2. Affinity Grid preparation of ribosomal complexes(a) Image of negatively stained ribosomal complexes adsorbed to a 2% CC Affinity Grid fromE. coli cell lysate containing 60 mM imidazole. (b) Image of vitrified ribosomal complexesadsorbed to a 20% HC Affinity Grid from the same E. coli cell lysate. The image shows mRNAemanating from the ribosomal complexes. The arrowheads point to 30S ribosomal subunits.(c) Image of vitrified ribosomal complexes prepared by lipid monolayer purification using a20% Ni-NTA lipid monolayer. No mRNA strands are visible. (d) Image of vitrified ribosomalcomplexes adsorbed to a 20% HC Affinity Grid from E. coli cell lysate that was treated withRNAse A. No mRNA strands are visible. Scale bar is 30 nm. (e, f) Illustrations of the twodifferent methods used to prepare Affinity Grids of ribosomal complexes. Either a drop of cellextract was pipetted onto an Affinity Grid (e) or the Affinity Grid was placed on top of cellextract contained in a well of a teflon block (f).Kelly et al. Page 13J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 3. 3D reconstructions of vitrified ribosomal complexes purified on Affinity Grids(a–d) 3D reconstruction of the 50S ribosomal subunit showing (a) representative classaverages, (b) the angular distribution plot, (c) the FSC curve indicating a resolution of 21 Åand (d) different views of the final density map with the fitted atomic model in red (pdb code:1ML5, chains a - x 2). (e–h) 3D reconstruction of the 30S ribosomal subunit showing (e)representative class averages, (f) the angular distribution plot, (g) the FSC curve indicating aresolution of 24 Å and (h) different views of the final density map with the fitted atomic modelin green (pdb code: 1ML5, chains A and C - X 2). (i–l) 3D reconstruction of the 70S ribosomeshowing (i) representative class averages, (j) the angular distribution plot, (k) the FSC curveindicating a resolution of 28 Å and (l) different views of the final density map with the fittedatomic model in blue (pdb code: 1ML5, all chains 2). The side length of individual panels in(a), (e) and (i) is 38 nm. The scale bar in (d) is 5 nm.Kelly et al. Page 14J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 4. Affinity Grid purification of His-tagged AQP9(a) Image of negatively stained Sf9 membrane extract. (b) Image of negatively stained AQP9purified by Ni-affinity and gel filtration chromatography. (c) Image of negatively stained AQP9adsorbed to a 2% CC Affinity Grid from Sf9 membrane extract containing 60 mM imidazole.Scale bar is 30 nm. (d, e) SimplyBlue stained SDS-PAGE gel (d) and Western blot (e) detectingHis-tagged AQP9 (lanes 1: AQP9 purified by conventional chromatographic methods, lanes2: AQP9 eluted from Affinity Grids). (f, g) Representative class averages of negatively stainedAQP9 purified by conventional chromatographic methods (f) or by adsorption to an AffinityGrid (g). Side length of individual panels is 26 nm.Kelly et al. Page 15J Mol Biol. Author manuscript; available in PMC 2009 October 3.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptKelly et al. Page 16Table 1Stability of Affinity Grids in the presence of detergents as assessed by visual inspection of the specimens in the electron microscope.Detergent CMC 0.5 min 1 min 2 min 5 min 10 min 20 min 30 min 60 min1% OG 0.53% *++ ++ ++ ++ ++ + + −2% OG ++ ++ ++ ++ ++ + − −3% OG + + + − − − − − − −4% OG − − − − − − − − − − − − −5% OG − − − − − − − − − − − − − − − −0.2% DM 0.087% *++ ++ ++ ++ ++ ++ + −0.02% DDM 0.0087% *++ ++ ++ ++ ++ ++ + −0.03% Triton X– 100 0.015% *++ ++ ++ + + − − − −0.014% Tween 20 0.0072% *+− − − − − − − − − − −0.5% CHAPS 0.49% *++ + + −− − − − − − − −0.2% Fos– Choline 11 0.062% *+ + + + + − − − −0.1% Digitonin 0.075% ** −− − − − − − − − − − − − − −*according to Anatrace Inc. (Maumee, OH); **according to Sigma Chemical Company (St. Louis, MO)++No apparent degradation of the lipid monolayer (continuous lipid monolayer)+Minor degradation of the lipid monolayer (appearance of some small holes in the lipid monolayer and of some small lipid vesicles)−Major degradation of the lipid monolayer (presence of many small holes in the lipid monolayer and of many small lipid vesicles)− −Almost complete degradation of the lipid monolayer (large grid areas without lipid monolayerJ Mol Biol. Author manuscript; available in PMC 2009 October 3. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptKelly et al. Page 17Table 2Stability of Affinity Grids in the presence of glycerol as assessed by visual inspection of the specimens in the electronmicroscope.% Glycerol 1 min 5 min 10 min 15 min1++ ++ + −2++ ++ + −3++ + + −4++ + − −−5+ + − −−++No apparent degradation of the lipid monolayer (continuous lipid monolayer)+Minor degradation of the lipid monolayer (appearance of some small holes in the lipid monolayer and of some small lipid vesicles)−Major degradation of the lipid monolayer (presence of many small holes in the lipid monolayer and of many small lipid vesicles)−−Almost complete degradation of the lipid monolayer (large grid areas without lipid monolayer)J Mol Biol. Author manuscript; available in PMC 2009 October 3. Citations (54)References (14)... Two different strategies, controlled liquid deposition 1,2 and grid surface modification, 3−14 have been pursued to improve the workflow and reliability of grid preparation. Initial surface modification approaches utilized grids coated with metal chelating lipids, 7,8 originally developed for the 2D crystallization of histidine-tagged proteins, 15 although the materials initially chosen led to preferred orientation problems when applied to cryo-EM sample preparation. 12 Subsequent efforts aimed at the specific capture of tagged protein targets have used nitrilotriacetic-acid (NTA)−polyethylene glycol (PEG) lipids, 5 grids coated with 2D streptavidin crystals on biotinylated lipid layers for biotinylated protein binding, 6 graphene oxide/NTA-modified graphene oxide grids, 4,9,11 and carbon nanomembranes. ...Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy StructurePreprintApr 2021 Md Rejaul HoqFrank S. Vago Kunpeng LiDavid H ThompsonRecent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å. These results demonstrate that the noncovalent capture of protein targets on EM grids modified with high-affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.ViewShow abstract... A number of different types of affinity grid have been introduced in recent years. One of these is based on Ni-NTA lipid monolayers that are picked up on holey-carbon films (Benjamin et al., 2016;Kelly et al., 2010;Kelly et al., 2008). Another is based on chemical functionalization that can be performed on oxidized, continuous carbon films (Llaguno et al., 2014). ...Monolayer-Crystal Streptavidin Support Films Provide an Internal Standard of cryo-EM Image QualityPreprintDec 2016 Bong-Gyoon HanZoe Watson Jamie H D CateRobert M. GlaeserAnalysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 Å, while the resolution of the map reached 4.0 Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4 Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 Å, a marked improvement over the value of 3.9 Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.ViewShow abstract... 31,32 While work on sample preparation methods for investigating fractionated or whole-cell lysates is ongoing, there already exist many approaches that can be used to reduce the complexity or target specific molecules from a mixture. Modified grid surfaces have been used for capturing proteins by His-tag, 39,40 biotin, 41 and antibody affinity. 42 These approaches can alleviate the need for purification, target low-abundance proteins, help with orientation bias, and be readily integrated in combination with clonal sets such as the ASKA library. ...Structural Biology in the Multi-Omics EraArticleFull-text availableMar 2020J CHEM INF MODELCaitlyn L. McCaffertyEric J. Verbeke Edward Marcotte David W TaylorRapid developments in cryo-electron microscopy have opened new avenues to probe the structures of protein assemblies in their near native states. Recent studies have begun applying single particle analysis to heterogeneous mixtures, revealing the potential of structural-omics approaches that combine the power of mass spectrometry and electron microscopy. Here, we highlight advances and challenges in sample preparation, data processing, and molecular modeling for handling increasingly complex mixtures. Such advances will help structural-omics methods extend to cellular level models of structural biology.ViewShow abstract... Examples include high-throughput grid preparation (33)(34)(35), time-resolved EM (36), and single-cell visual proteomics (14,15). Furthermore, antibody-functionalized EM grids were proposed to fish target proteins (37,38), and affinity grids designed to capture proteins with engineered his-tags (39). In both cases, the vitrified protein is supported by a continuous carbon film, which can limit the resolution obtained. ...Microfluidic protein isolation and sample preparation for high-resolution cryo-EMArticleFull-text availableJul 2019P NATL ACAD SCI USA Claudio Schmidli Stefan AlbiezLuca RimaThomas BraunHigh-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.ViewShow abstract... While in silico purification approaches will be useful, the rarer and more challenging the sample, the more necessary it will be to optimize sample purity biochemically; and as always, the adage garbage in, garbage out will usually apply. In this regard, one particularly attractive approach is the use of affinity grids for on-grid specimen purification (59)(60)(61). While the initial applications have been limited in scope (62)(63)(64)(65), it seems possible that a highly specific and well conjugated tag, coupled to a rigorous on-grid purification protocol, has the potential to provide a powerful means to isolate and explore rare biological assemblies with interesting functional properties. ...Challenges and Opportunities in Cryo-EM Single-Particle AnalysisArticleFull-text availableFeb 2019J BIOL CHEMDmitry LyumkisCryogenic electron microscopy (Cryo-EM), enables structure determination of macromolecular objects and their assemblies. Although the techniques have been developing for nearly 4 decades, they have gained widespread attention in recent years due to technical advances on numerous fronts, enabling traditional microscopists to break into the world of molecular structural biology. Many samples can now be routinely analyzed at near-atomic resolution using standard imaging and image analysis techniques. However, numerous challenges to conventional workflows remain, and continued technical advances open up entirely novel opportunities for discovery and exploration. Here, I will review some of the main methods surrounding cryo-EM with an emphasis specifically on single particle analysis (SPA), but will highlight challenges, open questions, and opportunities for methodology development.ViewShow abstract... Examples include high-throughput grid preparation (33)(34)(35), time-resolved EM (36), and single-cell visual proteomics (14,15). Furthermore, antibody-functionalized EM grids were proposed to fish target proteins (37,38), and affinity grids designed to capture proteins with engineered his-tags (39). In both cases, the vitrified protein is supported by a continuous carbon film, which can limit the resolution obtained. ...Microfluidic protein isolation and sample preparation for high resolution cryo-EMPreprintFull-text availableFeb 2019 Claudio Schmidli Stefan AlbiezLuca RimaThomas BraunHigh-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousands to a few million protein particles required for structure-determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than one microliter of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens new opportunities for the isolation of sensitive, endogenous protein complexes.ViewShow abstractFrom Tube to Structure: SPA Cryo-EM Workflow Using Apoferritin as an ExampleChapterMay 2021Meth Mol BiolChristoph A. DiebolderRebecca S. DillardLudovic RenaultIn this chapter, we present an overview of a standard protocol to achieve structure determination at high resolution by Single Particle Analysis cryogenic Electron Microscopy using apoferritin as a standard sample. The purified apoferritin is applied to a glow-discharged support and then flash frozen in liquid ethane. The prepared grids are loaded into the electron microscope and checked for particle spreading and ice thickness. The microscope alignments are performed and the data collection session is setup for an overnight data collection. The collected movies containing two-dimensional images of the apoferritin sample are then processed to obtain a high-resolution three-dimensional reconstruction.ViewShow abstractSmart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-MicroscopyArticleJun 2020ACS NANO Julian Scherr Zian Tang Maria Küllmer Andrey TurchaninTransmission electron cryo-microscopy (cryoEM) of vitrified biological specimens is a powerful tool for structural biology. Current preparation of vitrified biological samples starts off with sample isolation and purification, followed by the fixation in a freestanding layer of amorphous ice. Here, we demonstrate that ultrathin (∽10 nm) smart molecular nanosheets having specific bio-recognition sites embedded in a biorepulsive layer covalently bound to a mechanically stable carbon nanomembrane allow for a much simpler isolation and structural analysis. We characterize in detail the engineering of these nanosheets and their biorecognition properties employing complementary methods such as X-ray photoelectron and infrared spectroscopy, atomic force microscopy as well as surface plasmon resonance measurements. The desired functionality of the developed nanosheets is demonstrated by in situ selection of a His-tagged protein from a mixture and its subsequent structural analysis by cryoEM.ViewShow abstractWhat Could Go Wrong? A Practical Guide to Single-Particle Cryo-EM: From Biochemistry to Atomic ModelsArticleFeb 2020J CHEM INF MODELMichael A CianfroccoElizabeth Hua-Mei KelloggCryo-electron microscopy (cryo-EM) has enjoyed an explosive recent growth due to revolutionary advances in hardware and software, resulting in a steady stream of long-awaited, high-resolution structures with unprecedented atomic detail. With this comes an increased number of microscopes, cryo-EM facilities, and scientists eager to leverage the ability to determine protein structures without crystallization. However, numerous pitfalls and considerations beset the path towards high-resolution structures and are not necessarily obvious from literature surveys. Here, we detail the most common misconceptions when initiating a cryo-EM project, common technical hurdles as well as their solutions, and we conclude with a vision for the future of this exciting field.ViewShow abstractPreparation of Proteins and Macromolecular Assemblies for Cryo-electron MicroscopyChapterJan 2020Meth Mol Biol Lou Brillault Michael J LandsbergCryo-electron microscopy has become popular as the penultimate step on the road to structure determination for many proteins and macromolecular assemblies. The process of obtaining high-resolution images of a purified biomolecular complex in an electron microscope often follows a long, and in many cases exhaustive screening process in which many iterative rounds of protein purification are employed and the sample preparation procedure progressively re-evaluated in order to improve the distribution of particles visualized under the electron microscope, and thus maximize the opportunity for high-resolution structure determination. Typically, negative stain electron microscopy is employed to obtain a preliminary assessment of the sample quality, followed by cryo-EM which first requires the identification of optimal vitrification conditions. The original methods for frozen-hydrated specimen preparation developed over 40 years ago still enjoy widespread use today, although recent developments have set the scene for a future where more systematic and high-throughput approaches to the preparation of vitrified biomolecular complexes may be routinely employed. Here we summarize current approaches and ongoing innovations for the preparation of frozen-hydrated single particle specimens for cryo-EM, highlighting some of the commonly encountered problems and approaches that may help overcome these.ViewShow abstractShow moreStructure of the Escherichia coli ribosomal termination complex with release factor 2ArticleFull-text availableFeb 2003NATURE Bruno P KlaholzTillmann Pape Andrey V Zavialov Marin van HeelTermination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the SPF loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.ViewShow abstractExtraction of the reduced nicotinamide adenine dinucleotide phosphate. Δ 4 -3Ketosteroid5α-oxidoreductase of rat prostate with digitonin and potassium chlorideArticleJan 1974BIOCHEMISTRY-USRonald J. MooreJean D. WilsonViewExtraction of the reduced nicotinamide adenine dinucleotide phosphate:Δ4-3-ketosteroid-5α-oxidoreductase of rat prostate with digitonin and potassium chlorideArticleFeb 1974BIOCHEMISTRY-USR J MooreJ D Wilson5α-Reductase, the enzyme responsible for the conversion of testosterone to dihydrotestosterone in androgen-dependent target tissues, is a membrane-bound enzyme. In the ventral prostate of the rat its activity is found both in the nuclear membrane and in the membranes of the endoplasmic reticulum. Treatment of these membranes with digitonin (2 mg/mg of protein) plus 3 M KCl extracted the enzyme in a form that was retained on Bio-Gel A-1.5m column chromatography and failed to sediment during centrifugation at 100,000g for 1 hr. The activity in these extracts could be stabilized for as long as 4 days in the cold by either glycerol or NADPH. The pH optimum and apparent Km of the extracted enzymes were similar to those of the 5α-reductase in intact nuclei and microsomes. The 5α-reductase in the nuclear and microsomal extracts had an apparent mol wt of the order of 250,000-350,000 as estimated by gel filtration and a sedimentation coefficient of 13.5-15 S as estimated by density gradient centrifugation. The NADPH-stabilized enzyme was purified 90-fold.ViewShow abstractTwo-dimensional crystallization technique for imaging macromolecules, with application to antigen-antibody-complement complexesArticleFeb 1983NATUREE E UzgirisRoger D. KornbergTwo-dimensional crystals are formed from macromolecules bound on the surface of a lipid monolayer. A ligand linked to the lipid orientates the binding, and lateral diffusion of the lipids facilitates crystallization. The crystals are suitable for structural analysis by image processing of electron micrographs. An example is the formation of ordered arrays of antibodies on a monolayer of a lipid hapten, and subsequent decoration of these arrays with the first component of complement. Image processing indicates the arrangement of antibodies and the site of complement binding. This approach should be widely applicable to molecular complexes, such as those in replication, protein synthesis, hormone-receptor interaction and metabolic processes.ViewShow abstractSPIDER and WEB: Processing and Visualization of Images in 3D Electron Microscopy and Related FieldsArticleJan 1996J STRUCT BIOLJoachim FrankMichael Radermacher Pawel Penczek Ardean LeithThe SPIDER system has evolved into a comprehensive tool set for image processing, making use of modern graphics interfacing in the VMS and UNIX environment. SPIDER and WEB handle the complementary tasks of batch processing and visualization of the results. The emphasis of the SPIDER system remains in the area of single particle averaging and reconstruction, although a variety of other application areas have been added. Novel features are a suite of operations relating to the determination, modeling, and correction of the contrast transfer function and the availability of the entire documentation in hypertext format.ViewShow abstractDetermination of the fold of the core protein of hepatitis B virus by electron microscopyArticleApr 1997NATURE Bettina BöttcherS. A. and WynneR.A. CrowtherHepatitis B virus, a major human pathogen with an estimated 300 million carriers worldwide, can lead to cirrhosis and liver cancer in cases of chronic infection. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. The core protein, when expressed in bacteria, assembles into core shell particles, closely resembling the native core of the virus. Here we use electron cryomicroscopy to solve the structure of the core protein to 7.4 A resolution. Images of about 6,400 individual particles from 34 micrographs at different levels of defocus were combined, imposing icosahedral symmetry. The three-dimensional map reveals the complete fold of the polypeptide chain, which is quite unlike previously solved viral capsid proteins and is largely alpha-helical. The dimer clustering of subunits produces spikes on the surface of the shell, which consist of radial bundles of four long alpha-helices. Our model implies that the sequence corresponding to the immunodominant region of the core protein lies at the tip of the spike and also explains other properties of the core protein.ViewShow abstractEMAN: Semiautomated Software for High-Resolution Single-Particle ReconstructionsArticleJan 2000J STRUCT BIOL Steven J Ludtke Philip Rupert BaldwinWah ChiuWe present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN s extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data.ViewShow abstractTwo-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayerArticleJun 2001J MOL BIOL Luc LebeauFranck Lach Catherine Vénien-BryanCharles MioskowskiTwo-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.ViewShow abstractTwo-dimensional crystallization of membrane proteins: The lipid layer strategyArticleSep 2001FEBS LETTDaniel Lévy Mohamed ChamiJ L RigaudDue to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two-dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico-chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.ViewShow abstractAccurate determination of local defocus and specimen tilt in electron microscopyArticleJul 2003J STRUCT BIOL Joseph A Mindell Nikolaus GrigorieffAccurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively.ViewShow abstractShow moreAdvertisementRecommended publicationsDiscover moreArticleSynthesis of Transferrin-Mitomycin C Conjugate as a Receptor-Mediated Drug Targeting System.May 1996 · Biological Pharmaceutical Bulletin Tetsuro TanakaYoshiharu KaneoMasahide MIYASHITAMacromolecular conjugates of mitomycin C (MMC) were synthesized by binding an active ester of glutarylated MMC (MMC-G-OSu) to human holo-transferrin (TF). Water-soluble TF-MMC conjugates (TF-G-MMC) were obtained in a good yield ( 95%) by this method. The MMC content of the conjugate increased (0.82-9.49 MMC/w%) with increasing amounts of MMC-G-OSu added to the conjugation mixture. Sodium dodecyl ... [Show full abstract] sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed no aggregation in these conjugates. 125I-TF-G-MMC was bound specifically to the TF receptor on Sarcoma 180 cells; the measurement of equilibrium binding of the 125I-labeled conjugate resulted in a saturation isotherm. The amount of conjugate specifically bound to the TF receptor decreased as the MMC content of the conjugate increased. However, it was found that the conjugate with an MMC content below 10 mol MMC/mol TF still retains a binding activity of more than half that of TF. Therefore, when an optimal chemical modification was chosen, TF could be used as a tumor specific drug carrier.Read moreArticleMolecular characteristics of the transferrin-receptor complex of the rabbit reticulocyteJanuary 1978 · Journal of Supramolecular StructureHsiang-Yun Yang Hu Philip AisenA macromolecular complex of transferrin and a membrane component was isolated by gel filtration chromatography from Triton X-100-solubilized ghosts of reticulocytes previously incubated with 125I-labeled transferrin. This complex is believed to be transferrin specifically associated with its primary receptors. Following the procedures of Clark [14], the complex in Triton X-100 was found to behave ... [Show full abstract] as an asymmetric molecule with a molecular weight of approximately 250,000 and an axial ratio of 9:1. On SDS-poly-acrylamide gel electrophoresis the complex displays, in addition to transferrin, components of molecular weights 176,000 and 95,000, respectively. The larger component may be a dimer of the smaller. Each appears to crosslink, with dimethyl suberimidate, to transferrin. These results are compatible with the hypothesis that the transferrin receptor itself has a molecular weight near 175,000 and may be a dimer of two smaller components each of molecular weight near 95,000.Read moreArticleKilling of K562 cells with conjugates between human transferrin and a ribosome-inactivating protein...March 1988 · British Journal of Haematology Gaetano BergamaschiMario CazzolaL Dezza[...] Douglas LappiCellular iron uptake is mediated by binding of transferrin with specific surface receptors and internalization of the Fe-transferrin-receptor complex. This has been examined as a possible pathway for carrying into leukaemic cells a ribosome-inactivating protein (RIP), SO-6, derived from Saponaria officinalis. Purified human differic transferrin was conjugated with SO-6 and a pool of proteins was ... [Show full abstract] obtained, with variable numbers of SO-6 molecules linked to a single transferrin molecule. Human erythroleukaemic K562 cells were grown in the presence of human transferrin, SO-6 and human transferrin conjugated with SO-6. The conjugate was found to be internalized via binding with transferrin receptor. Whereas the presence of unconjugated human transferrin and SO-6 in the medium did not significantly influence K562 cell growth, the conjugated proteins displayed an inhibitory activity on cell proliferation. This was maximal after 72 h at a transferrin concentration of 10-9m, with about 50% of cells being killed. Bovine transferrin, present in fetal calf serum, did not appear to compete with human diferric transferrin in binding to K562 cells in suspension culture. In a clonogenic assay, colony formation by leukaemic cells was not influenced by free SO-6 or transferrin, whereas the conjugated proteins were markedly inhibitory (about 100% at 10-9m). Our findings indicate that SO-6 can be efficiently carried into mammalian cells via the transferrin-transferrin receptor cycle and exert its ribosome inactivating activity. This is in keeping with the existence of an alternative pathway of transferrin endocytosis in addition to the classic acidic endosome pathway. From a practical viewpoint, conjugates between transferrin and SO-6 can be useful tools for studying the expression of transferrin receptors, and deserve also to be investigated for a possible use in cancer therapy.Read moreArticleFull-text availableAltered Ferritin Subunit Composition: Change in Iron Metabolism in Lens Epithelial Cells and Downstr...September 2010 · Investigative Ophthalmology Visual ScienceJ HarnedJenny B. FerrellMarilyn M Lall[...] Mary McgahanThe iron storage protein ferritin is necessary for the safe storage of iron and for protection against the production of iron-catalyzed oxidative damage. Ferritin is composed of 24 subunits of two types: heavy (H) and light (L). The ratio of these subunits is tissue specific, and alteration of this ratio can have profound effects on iron storage and availability. In the present study, siRNA for ... [Show full abstract] each of the chains was used to alter the ferritin H:L chain ratio and to determine the effect of these changes on ferritin synthesis, iron metabolism, and downstream effects on iron-responsive pathways in canine lens epithelial cells. Primary cultures of canine lens epithelial cells were used. The cells were transfected with custom-made siRNA for canine ferritin H- and L-chains. De novo ferritin synthesis was determined by labeling newly synthesized ferritin chains with 35S-methionine, immunoprecipitation, and separation by SDS-PAGE. Iron uptake into cells and incorporation into ferritin was measured by incubating the cells with 59Fe-labeled transferrin. Western blot analysis was used to determine the presence of transferrin receptor, and ELISA was used to determine total ferritin concentration. Ferritin localization in the cells was determined by immunofluorescence labeling. VEGF, glutathione secretion levels, and cystine uptake were measured. FHsiRNA decreased ferritin H-chain synthesis, but doubled ferritin L-chain synthesis. FLsiRNA decreased both ferritin H- and L-chain synthesis. The degradation of ferritin H-chain was blocked by both siRNAs, whereas only FHsiRNA blocked the degradation of ferritin L-chain, which caused significant accumulation of ferritin L-chain in the cells. This excess ferritin L-chain was found in inclusion bodies, some of which were co-localized with lysosomes. Iron storage in ferritin was greatly reduced by FHsiRNA, resulting in increased iron availability, as noted by a decrease in transferrin receptor levels and iron uptake from transferrin. Increased iron availability also increased cystine uptake and glutathione concentration and decreased nuclear translocation of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium. Most of the effects of altering the ferritin H:L ratio with the specific siRNAs were due to changes in the availability of iron in a labile pool. They caused significant changes in iron uptake and storage, the rate of ferritin synthesis and degradation, the secretion of VEGF, and the levels of glutathione in cultured lens epithelial cells. These profound effects clearly demonstrate that maintenance of a specific H:L ratio is part of a basic cellular homeostatic mechanism.View full-textDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleNo account? Sign upCompanyAbout usNewsCareersSupportHelp CenterBusiness solutionsAdvertisingRecruiting© 2008-2021 ResearchGate GmbH. 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