Seeing GroEL at 6 Å Resolution by Single Particle Electron Cryomicroscopy

Ludtke, Steven J.; Chen, Dong Hua; Song, Jiu Li; Chuang, David T.; Chiu, Wah

doi:10.1016/j.str.2004.05.006

Cited by 186 publications

(203 citation statements)

References 35 publications

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“…Fourier power spectra for each micrograph displayed first minima in the range of 19 to 21 Å. A data set of ;3400 particles was compiled using "boxer" of the EMAN software package (Ludtke et al, 2004). Further processing was performed using Imagic-5 (Image Science) at a sampling frequency of 2.5 Å/pixel on the specimen scale.…”

Section: Single-particle Analysismentioning

confidence: 99%

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

Boehm

Krynická

et al. 2012

The Plant Cell

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FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)-tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2 2 mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.

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Section: Single-particle Analysismentioning

confidence: 99%

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

Boehm

Krynická

et al. 2012

The Plant Cell

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“…As a second indicator, we measured the cross-correlation coefficient of the average map obtained at each iteration with a 6 Å map of GroEL, filtered to 20 Å obtained from single-particle analysis by Ludtke et al (2004). As the average map gets progressively better with the iterations, this quantity is expected to increase.…”

Section: Validation With Groelmentioning

confidence: 99%

Classification and 3D averaging with missing wedge correction in biological electron tomography

Bartesaghi

Sprechmann

Liu

et al. 2008

Journal of Structural Biology

168

209

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Strategies for the determination of 3D structures of biological macromolecules using electron crystallography and single-particle electron microscopy utilize powerful tools for the averaging of information obtained from 2D projection images of structurally homogeneous specimens. In contrast, electron tomographic approaches have often been used to study the 3D structures of heterogeneous, one-of-a-kind objects such as whole cells where image-averaging strategies are not applicable. Complex entities such as cells and viruses, nevertheless, contain multiple copies of numerous macromolecules that can individually be subjected to 3D averaging. Here we present a complete framework for alignment, classification, and averaging of volumes derived by electron tomography that is computationally efficient and effectively accounts for the missing wedge that is inherent to limited-angle electron tomography. Modeling the missing data as a multiplying mask in reciprocal space we show that the effect of the missing wedge can be accounted for seamlessly in all alignment and classification operations. We solve the alignment problem using the convolution theorem in harmonic analysis, thus eliminating the need for approaches that require exhaustive angular search, and adopt an iterative approach to alignment and classification that does not require the use of external references. We demonstrate that our method can be successfully applied for 3D classification and averaging of phantom volumes as well as experimentally obtained tomograms of GroEL where the outcomes of the analysis can be quantitatively compared against the expected results.

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“…The MM values obtained for solutions of unliganded GltS at low ionic strength (Table 1, lines 1-4) indicate that Ͼ90% of the protein is present as an (␣␤) 6 hexamer. GltS solutions that had been preincubated for up to 16 h with the L-Gln analog MetS (1 mM) and the GltS substrate 2-OG (1 mM), alone (not shown) or in combination, yielded similar R g and MM values (Table 1, lines [17][18][19][20]. On the contrary, preincubation with NADP ϩ either alone or in combination with MetS and 2-OG (Table 1, lines 21-24 and 13-16) resulted in a decrease of both R g and MM, suggesting a shift in the oligomeric equilibrium toward smaller particles.…”

Section: Saxs and Stoichiometry Of The Nadph-glts Complex In Solutionmentioning

confidence: 99%

“…The combined use of cryo-EM and SAXS allowed us to obtain an electron density map of the GltS (␣␤) 6 hexamer at a subnanometer resolution, which has been reached only recently, and only in a few cases, for cryo-EM-based structure determinations (17)(18)(19)(20)(21)(22). Moreover, we propose a homology model of ␤GltS, and, more importantly, that of the ␣␤ protomer.…”

mentioning

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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Seeing GroEL at 6 Å Resolution by Single Particle Electron Cryomicroscopy

Cited by 186 publications

References 35 publications

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

Classification and 3D averaging with missing wedge correction in biological electron tomography

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

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