Appion: An integrated, database-driven pipeline to facilitate EM image processing

Lander, Gabriel C.; Stagg, Scott M.; Voss, Neil R.; Cheng, Anchi; Fellmann, D.; Pulokas, James; Yoshioka, Craig; Irving, Christopher; Mulder, Anke M.; Lau, Pick Wei; Lyumkis, Dmitry; Potter, Clinton S.; Carragher, Bridget

doi:10.1016/j.jsb.2009.01.002

Cited by 890 publications

(772 citation statements)

References 27 publications

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“…Initial processing was performed using the NRAMM Appion pipeline (46). Particles were picked automatically using DoG (46), and contrast-transfer-function parameters were calculated using ACE (47).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Images were collected at 52,000× magnification with a −1.3 μm defocus for a final magnified pixel size of 2.05 Å/pix. Both Automated particle picking performed with DoG-Picker 28 and reference-free 2-D classification with iterative MRA-MSA 29 were executed through the Appion database 30 . For 3D analysis, micrographs were first CTF estimated with GCTF then particles were extracted, phase-flipped, and subjected to reference-based 3D classification and refinement in Relion version 2.0 31 .…”

Section: Methods/supplementary Informationmentioning

confidence: 99%

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows

Sok

Vadnais

et al. 2017

Nature

152

198

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No immunogen to date has reliably elicited broadly neutralizing antibodies (bnAbs) to HIV in humans or animal models. Advances in the design of immunogens (BG505 SOSIP) that antigenically mimic the HIV envelope glycoprotein (Env)1 have improved the elicitation of potent isolate-specific Ab responses in rabbits2 and macaques3, but so far failed to induce bnAbs. One possible contributor to this failure is that the relevant antibody repertoires are poorly suited to target somewhat occluded conserved epitope regions on Env relative to exposed variable epitopes. To test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach over 70 amino acids in length4–9. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody (mAb) isolated from this cow harbored an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 μg/ml. We note that breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

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“…Images were collected using the Leginon data collection software (48) on a Fei Tecnai F20 microscope using a 80,000× magnification (1.5 Å/pixel) in low-dose mode (20e − /Å 2 ) on a Gatan 4K × 4K pixel CCD camera (15-μm pixel size). The 2D data were processed using the Appion package (49).…”

Section: Methodsmentioning

confidence: 99%

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

Querol-Audí

Cheng

et al. 2012

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

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Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.F lap endonuclease 1 (FEN1) belongs to a class of essential nucleases (the FEN1 5′ nuclease superfamily) present in all domains of life (1). FEN1 catalyzes the endonucleolytic cleavage of bifurcated DNA or RNA structures known as 5′ flaps. These 5′ flaps are generated during lagging strand DNA synthesis or during long-patch base excision repair. The FEN1 substrates are in fact double-flap DNA (dfDNA) with DNA on the opposite side of the 5′ flap, forming a single nucleotide 3′ flap when bound to the enzyme (2, 3). By removing the 5′ ssDNA or RNA flap from such substrates, FEN1 produces a single nicked product that could be sealed by the subsequent action of a DNA ligase (4). Consistent with its crucial role in DNA replication and repair, FEN1 is highly expressed in all proliferative tissues, and its activity is key for the maintenance of genomic integrity (5). FEN1 has been identified as a cancer susceptibility gene, and mutations in it have been linked to a number of genetic diseases, such as EM map myotonic dystrophy, Huntington disease, several ataxias, fragile X syndrome, and cancer (6-10).The nuclease activity of FEN1 can be stimulated by association with processivity clamps such as proliferating cell nuclear antigen (PCNA), which encircle DNA at sites of replication and repair (11)(12)(13). PCNA is a recognized master coordinator of cellular responses to DNA damage and interacts with numerous DNA repair and cell-cycle control proteins. In this capacity, PCNA serves not only as a mobile platform for the attachment of these proteins to DNA but, importantly, plays an active role in the recru...

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Appion: An integrated, database-driven pipeline to facilitate EM image processing

Cited by 890 publications

References 27 publications

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

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

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

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