Negative staining and image classification — powerful tools in modern electron microscopy

Ohi, Melanie D.; Liu, Ying; Cheng, Yifan; Walz, Thomas

doi:10.1251/bpo70

Cited by 639 publications

(601 citation statements)

References 33 publications

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“…Rubisco-RLSMT complexes mixed at different ratios were prepared for electron microscopy by negative staining with uranyl formate as described in ref. 29. For cryo-EM, Rubisco-RLSMT mixtures at a molar ratio of 1:1 were applied to holey carbon grids and frozen in liquid ethane.…”

Section: Methodsmentioning

confidence: 99%

Rubisco in complex with Rubisco large subunit methyltransferase

Raunser

Magnani

Huang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplastlocalized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-Å density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.electron microscopy ͉ LSMT ͉ SET domain ͉ single particle S ET [SU(VAR)3-9, E(Z), and TRX] domain protein lysine methyltransferases (PKMTs) are a structurally unique class of enzymes that catalyze the formation of site-specific methylated lysine residues in a number of different polypeptide substrates including cytochrome c (1, 2), histones (3), ribosomal proteins (3, 4), p53 (5), TAF10 (6), ␥-tocopherol methyltransferase (7), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (8). All SET domain PKMTs have a unique and conserved structural motif that contains separate binding sites for the target protein substrate and the methyl donor, Sadenosylmethionine (AdoMet) (9). The mechanisms by which these enzymes achieve site-specific methylation is of great interest and histone-specific SET domain protein methyltransferases (HKMTs) have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers (9, 10). The specificity of several SET domain PKMTs has been examined through structural and biochemical analyses of ternary complexes with bound polypeptide substrates, but all of these studies used short synthetic polypeptide mimetics of the intact polypeptide substrate and therefore do not account for the potential influence of areas outside the immediate residues flanking the target lysine methylation site (11)(12)(13)(14)(15)(16). Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco (8,17), an essential photosynthetic enzyme with a hexadecameric structure and large molecular mass (Ϸ534 kDa). Detailed information is available regarding the structure, catalytic mechanism, active site residues, and the kinetic reaction mechanism for pea RLSMT (7,8,17,18)...

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Section: Methodsmentioning

confidence: 99%

Rubisco in complex with Rubisco large subunit methyltransferase

Raunser

Magnani

Huang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…48 In brief, purified protein samples were adsorbed to glow discharged carbon-coated copper grids (Ted Pella Inc.., G400), stained with 0.75% (w/v) uranyl formate, and air dried. Raw images were recorded at a nominal magnification of 49,000x on a 4K x 4K Eagle charge-coupled device (CCD) camera (FEI, Hillsboro, USA) with a Tecnai Spirit transmission electron microscope (FEI, Hillsboro, USA) operated at an accelerating voltage of 120 kV.…”

Section: Electron Microscopymentioning

confidence: 99%

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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(2015) Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms, Autophagy, 11:6, 891-905, DOI: 10.1080/15548627.2015 The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1 [CTD]) and Atg13 (Atg13 [CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1 [CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

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“…No further purification was required, since excess unbound CAP (47 kDa) and multiRNAPcontaining aggregates (Ͼ1,000 kDa) are readily distinguished from CAP-RNAP-promoter complexes (500 kDa) in EM images. Complexes were visualized in a carbon-sandwich uranyl formate negative-stain preparation, as described by Ohi et al (20). Even though DNA has weaker contrast than protein in negative stain (21)(22)(23)(24), this preparation method has yielded the most structural detail thus far of the complex.…”

Section: Design Assembly and Imaging Of A Class I Cap-rnap-promotermentioning

confidence: 99%

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

Hudson

Quispe

Lara-González³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions ؊78 to ؉20 of a Class I CAP-dependent promoter with a CAP site at position ؊61.5 and a premelted transcription bubble. A 20-Å electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbonsandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP ␣ subunit C-terminal domain (␣CTD), interactions of ␣CTD with 70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNAbend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP ␤ , ␤, and 70 subunits.catabolite activator protein ͉ deoxyribonucleic acid ͉ RNA polymerase holoenzyme ͉ gene regulation ͉ electron microscopy

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Negative staining and image classification — powerful tools in modern electron microscopy

Cited by 639 publications

References 33 publications

Rubisco in complex with Rubisco large subunit methyltransferase

Rubisco in complex with Rubisco large subunit methyltransferase

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

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