Visualizing density maps with UCSF Chimera

Goddard, Thomas D.; Huang, Conrad C.; Ferrin, Thomas E.

doi:10.1016/j.jsb.2006.06.010

Cited by 837 publications

(799 citation statements)

References 13 publications

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“…To perform focused refinement of the connector protein region, we low‐pass filtered the refined baseplate map to 80 Å and generated a small mask around TssK region with Segger (Pintilie et al , 2010) in UCSF Chimera (Pettersen et al , 2004; Goddard et al , 2007). Mask was prepared for RELION2.1 with relion_mask_create with extended width and soft edge of five pixels.…”

Section: Methodsmentioning

confidence: 99%

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

et al. 2017

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The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath–tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath–tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non‐contractile Vibrio cholerae sheaths by cryo‐electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F2/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.

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Section: Methodsmentioning

confidence: 99%

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

et al. 2017

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“…To perform the fitting of atomic data into the cryo-EM map, the following programs were compared: (i) rigid body fitting in Fourier space with Colores and Colacor algorithms of SITUS (46,47); (ii) real space fitting with the FoldHunterP algorithm of EMAN (48,49); (iii) real space fitting using the operation "Fit Model in Map" of Chimera (50,51). A first ␣ 2 dimer was fitted, and the 3-fold symmetry was used to calculate the atomic coordinates of the other two.…”

Section: Methodsmentioning

confidence: 99%

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Cottevieille

Larquet

Jonić

et al. 2008

Journal of Biological Chemistry

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The three-dimensional structure of the hexameric (␣␤) 6 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex ironsulfur flavoprotein. In the hexameric species, interprotomeric ␣-␣ and ␣-␤ contacts are mediated by the C-terminal domain of the ␣ subunit, which is based on a ␤ helical fold so far unique to glutamate synthases. The ␣␤ protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the ␤ subunit, to the FMN on the ␣ subunit, through the low potential [4Fe-4S] 1؉/2؉ centers on the ␤ subunit and the [3Fe-4S] 0/1؉ cluster on the ␣ subunit. The (␣␤) 6 hexamer exhibits a concentration-dependent equilibrium with ␣␤ monomers and (␣␤) 2 dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP ؉ . Hexamerization seems to decrease the catalytic efficiency of the ␣␤ protomer only 3-fold by increasing the K m values measured for L-Gln and 2-OG. However, it cannot be ruled out that the (␣␤) 6 hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.Glutamate synthases (GltS) 2 are complex iron-sulfur flavoproteins that catalyze the reductive transfer of the L-Gln amide group to the C2 carbon of 2-oxoglutarate (2-OG), yielding two molecules of L-glutamate (L-Glu). GltS are found in bacteria, yeast, and plants, where they form with glutamine synthetase an essential pathway for ammonia assimilation (1-3). Bacterial NADPH-dependent GltS (NADPH-GltS) is formed by one ␣ subunit (␣GltS) and one ␤ subunit (␤GltS) of 162 and 52 kDa, respectively, for the Azospirillum brasilense GltS. The ␣␤ protomer contains one FAD (on ␤GltS), one FMN (on ␣GltS), and three different iron-sulfur clusters (one [3Fe-4S] 0/1ϩ cluster on ␣GltS and two [4Fe-4S] 1ϩ/2ϩ centers on ␤GltS; Fig. 1A). On the basis of sequence, structural, and mechanistic similarities, the NADPH-GltS serves as a model for the other two main forms of GltS, namely (i) the ferredoxin-dependent GltS found in cyanobacteria and photosynthetic tissues of plants, which is similar to ␣GltS and (ii) the eukaryotic type of GltS, which is NADH-dependent and is found in yeast, nonphotosynthetic tissues of plants, and lower eukaryotes; this GltS species is formed by a single polypeptide chain derived from the fusion of bacterial ␣ and ␤ subunits.Several lines of evidence support an essential role of GltS in all of these cell types, making it a potential target of novel drugs. For example, the NADPH-GltS has been found to be essential in Mycobacterium tuberculosis, where it has been shown that Ͻ1 M azaserine, a GltS inhibitor, is sufficient t...

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“…Figures were prepared with Chimera [11]. The PCNA-PolB1-Fen1-DNA structure was solved using the EMD-5220 map [12] as a starting model and refined with EMAN using 6878 images sampled at 6.4 Å/pixel to a resolution of ∼30 Å.…”

Section: Electron Microscopymentioning

confidence: 99%

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

Cannone

Beattie

et al. 2015

Biochemical Journal

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DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNApolymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.

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Visualizing density maps with UCSF Chimera

Cited by 837 publications

References 13 publications

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

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