GraFix: sample preparation for single-particle electron cryomicroscopy

Kastner, Berthold; Fischer, N.; Golas, Monika M.; Sander, Bjoern; Dube, Prakash; Boehringer, Daniel; Hartmuth, Klaus; Deckert, Jochen; Hauer, Florian; Wolf, Elmar; Uchtenhagen, Hannes; Urlaub, Henning; Herzog, Franz; Peters, Jan Michael; Poerschke, D.; Lührmann, Reinhard; Stark, Holger

doi:10.1038/nmeth1139

Cited by 523 publications

(548 citation statements)

References 16 publications

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“…The DNA probes were preannealed and contained the response element for gene GADD45 flanked by random nonspecific sequences. Tetramers of p53 bound to DNA were isolated using GraFix methodology (29). Selected fractions were adsorbed on carbon coated grids and stained with uranyl formate in double carbon sandwich.…”

Section: Methodsmentioning

confidence: 99%

“…Fulllength p53 (flp53) was allowed to bind DNA sequences containing the GADD45 response element, a specific target for p53. A pure population of flp53 tetramers bound to DNA was isolated and crosslinked by a glycerol-glutaraldehyde combined gradient, a method known as GraFix (29). The procedure facilitates structural studies of fragile complexes and does not generally promote significant structural differences or artifacts in tested samples.…”

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confidence: 99%

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Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA

Melero

Rajagopalan

Lázaro

et al. 2010

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

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The multidomain homotetrameric tumor suppressor p53 has two modes of binding dsDNA that are thought to be responsible for scanning and recognizing specific response elements (REs). The C termini bind nonspecifically to dsDNA. The four DNA-binding domains (DBDs) bind REs that have two symmetric 10 base-pair sequences. p53 bound to a 20-bp RE has the DBDs enveloping the DNA, which is in the center of the molecule surrounded by linker sequences to the tetramerization domain (Tet). We investigated by electron microscopy structures of p53 bound to DNA sequences consisting of a 20-bp RE with either 12 or 20 bp nonspecific extensions on either end. We found a variety of structures that give clues to recognition and scanning mechanisms. The 44-and 60-bp sequences gave rise to three and four classes of structures, respectively. One was similar to the known 20-bp structure, but the DBDs in the other classes were loosely arranged and incompatible with specific DNA recognition. Some of the complexes had density consistent with the C termini extending from Tet to the DNA, adjacent to the DBDs. Single-molecule fluorescence resonance energy transfer experiments detected the approach of the C termini towards the DBDs on addition of DNA. The structural data are consistent with p53 sliding along DNA via its C termini and the DNA-binding domains hopping on and off during searches for REs. The loose structures and posttranslational modifications account for the affinity of nonspecific DNA for p53 and point to a mechanism of enhancement of specificity by its binding to effector proteins.protein | recognition | specificity

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA

Melero

Rajagopalan

Lázaro

et al. 2010

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

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“…40 As a proof of concept, we subjected purified Atg17-Atg31-Atg29 to GraFix treatment and then analyzed the treated complex by negative-stain EM. We found that GraFix treatment did not result in aggregation of the highly extended Atg17-Atg31-Atg29 subcomplex, and 2-dimensional (2D) image analysis further showed that the limited glutaraldehyde crosslinking did not alter the overall structure of this subcomplex (Fig.…”

Section: Visualizing the Minimal Atg1 Complex By Electron Microscopymentioning

confidence: 99%

“…40 The purified complex was first concentrated using an Amicon 10K cutoff concentrator. The concentrated sample was then overlaid on top of a tube containing a linear 12-24% glycerol (BioShop, GLY004) gradient and a glutaraldehyde gradient (0 to 0.05%; Sigma-Aldrich, G7651) in buffer (150 mM NaCl, 50 mM HEPES, pH 8.0).…”

Section: Gradient Fixationmentioning

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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“…A gel filtration fraction corresponding to the RECQ1 tetramer was further processed by means of a glycerol and glutaraldehyde gradient [GraFix (31)] to stabilize fragile, existing protein/protein interfaces gently. The GraFix treatment was essential to obtain negative-stain EM preparations tractable by single-particle analysis, whereas a noncross-linked sample resulted in unresolvable heterogeneity.…”

mentioning

confidence: 99%

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

Pike

Gomathinayagam

Swuec

et al. 2015

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

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RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.DNA helicases | RecQ | genome stability | Holliday junction | fork reversal R ecQ helicases are a family of ATP-dependent motor proteins that play central roles in maintaining genome stability. Defects in three of the five human RecQ homologs give rise to distinct genetic disorders associated with genomic instability, cancer predisposition, and premature aging (1-5). The unique clinical features of these disorders support the notion that the different RecQ helicases have nonoverlapping functions, but the molecular basis for their different enzymatic activities remains unclear. RecQ helicases catalyze ATP-dependent DNA unwinding in the 3′-5′ direction. Additionally, members of this helicase family have been shown to tackle an unparalleled breath of noncanonical DNA structures, such as fork DNA, G-quadruplexes, D-loops, and Holliday junction (HJ) structures (6-8). However, our understanding of the physical mechanisms by which RecQ helicases recognize and process their physiological substrates remains remarkably limited.RECQ1 is the shortest of the human RecQ-family helicases, comprising the bipartite ATPase domain common to all superfamily 2 (SF2) helicases, the RecQ-specific C-terminal domain (RQC), and short extensions on the N and C termini. We recently discovered a specific function of RECQ1 in branch migration and restart of reversed DNA replication forks upon DNA topoisomerase I inhibition that is not shared by other human RecQ helicases, such as Werner (WRN) or Bloom (BLM) syndrome proteins (9). On the other hand, BLM is the sole human RecQ helicase member specifically able to resolve double-HJ junction structures in conjunction with DNA topoisomerase III alpha and the RMI1 and RMI2 accessory proteins (10-12). These findings lead us to hypothesize that the sp...

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GraFix: sample preparation for single-particle electron cryomicroscopy

Cited by 523 publications

References 16 publications

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA

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

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

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