SPARX, a new environment for Cryo-EM image processing

Hohn, Michael H.; Tang, Grant; Goodyear, Grant; Baldwin, Philip R.; Huang, Zhong; Penczek, Pawel A.; Yang, Chao; Glaeser, Robert M.; Adams, Paul D.; Ludtke, Steven J.

doi:10.1016/j.jsb.2006.07.003

Cited by 364 publications

(332 citation statements)

References 20 publications

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“…Individual CCD frames were normalized and Weiner filtered with the Appion processing package (29), and 18,761 individual particle images were automatically selected (30). Individual particle images were analyzed using the SPIDER and SPARX EM image processing packages (31,32).…”

Section: Methodsmentioning

confidence: 99%

A soluble α-synuclein construct forms a dynamic tetramer

Wang

Perovic

Chittuluru

et al. 2011

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

418

522

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A heterologously expressed form of the human Parkinson diseaseassociated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 Nterminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three-or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. T he protein α-synuclein (αSyn) is associated with the two most prevalent neurodegenerative diseases, Parkinson disease (PD) and Alzheimer's disease (AD). The presence of αSyn-rich aggregates (Lewy bodies) in neurons of the substantia nigra is the defining histopathological hallmark of PD, and is used to differentiate PD from other neurological disorders (1). Monogenic point mutations (A30P, A53T, and E46K) as well as gene duplication and triplication of the αSyn locus have been identified as causal factors of early onset familial PD; E46K has also been associated with Lewy body dementia, the second most common form of dementia after AD (2-4).αSyn is small (140 residues), and though the C-terminal region (∼residues 100-140) is highly acidic and expected to be disordered, the first 100 residues are predicted to be structured and to have α-helical propensity (SI Appendix, Fig. S1). Stable helical structures have been detected by circular dichroism (CD) and NMR when αSyn is incubated with detergent micelles and lipid vesicles (5, 6). Soluble αSyn is typically referred to as an "intrinsically disordered" protein (7,8). However, we herein report the biophysical characterization of a purified soluble form of αSyn that is oligomeric and fractionally occupies helical structures in the absence of micelles or vesicles. The αSyn construct used in our work is purified by use of an N-terminal GST affinity tag under mild conditions to preserve any native structure. After removal of the GST tag, a 10-residue N-terminal extension remains on the αSyn. However, the similarity of the 1 H, 15 N heteronuclear single-quantum coherence (HSQC) fingerprint of our αSyn construct (SI Appendix, Figs. S2 and S3) to those reported by other groups for αSyn suggests that the N-terminal extension does not change structural tendencies significantly. The αSyn construct described here is not toxic to membranes or cells, does not readily aggregate or ...

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Section: Methodsmentioning

confidence: 99%

A soluble α-synuclein construct forms a dynamic tetramer

Wang

Perovic

Chittuluru

et al. 2011

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

418

522

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“…Images of negatively stained specimens were processed by using the Spider software package (31). Images of vitrified complexes were processed by using SPARX (32). The mixture of asymmetric and D4-symmetric objects required a protocol specifically designed to determine the 3D structure of the Rubisco-RLSMT complex (see flow chart depicted in Fig.…”

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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“…We conducted our tests using simulated data within the environment of the SPARX single particle package (http://macro-em.org/sparxwiki) [31]. All methods were tested on a Linux workstation equipped with two AMD 2.4 GHz CPUs and 8GB of memory.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, here we focused on simple 2-D and 3-D image registration tasks and on commonly used in EM linear and quadratic interpolations and compared them with gridding-based interpolation. Future work on optimum alignment methods should be facilitated by the release of the SPARX system [31]. By promoting seamless access to multiple software packages it encourages standardization of test data within the community and simplifies testing.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM image alignment based on nonuniform fast Fourier transform

Yang

Penczek

2008

Ultramicroscopy

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In single particle analysis, two-dimensional (2-D) alignment is a fundamental step intended to put into register various particle projections of biological macromolecules collected at the electron microscope. The efficiency and quality of three-dimensional (3-D) structure reconstruction largely depends on the computational speed and alignment accuracy of this crucial step. In order to improve the performance of alignment, we introduce a new method that takes advantage of the highly accurate interpolation scheme based on the gridding method, a version of the nonuniform Fast Fourier Transform, and utilizes a multi-dimensional optimization algorithm for the refinement of the orientation parameters. Using simulated data, we demonstrate that by using less than half of the sample points and taking twice the runtime, our new 2-D alignment method achieves dramatically better alignment accuracy than that based on quadratic interpolation. We also apply our method to image to volume registration, the key step in the single particle EM structure refinement protocol. We find that in this case the accuracy of the method not only surpasses the accuracy of the commonly used real-space implementation, but results are achieved in much shorter time, making griddingbased alignment a perfect candidate for efficient structure determination in single particle analysis.

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SPARX, a new environment for Cryo-EM image processing

Cited by 364 publications

References 20 publications

A soluble α-synuclein construct forms a dynamic tetramer

A soluble α-synuclein construct forms a dynamic tetramer

Rubisco in complex with Rubisco large subunit methyltransferase

Cryo-EM image alignment based on nonuniform fast Fourier transform

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