High-resolution structure of proteinase K cocrystallized with digalacturonic acid

Larson, Steven B.; Day, John; Nguyen, Chieugiang; Cudney, Robert; McPherson, Alexander

doi:10.1107/s1744309109002218

Cited by 5 publications

(4 citation statements)

References 17 publications

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“…Two synthetic cases were chosen with different sizes and symmetries. Proteinase K (PK) is a 30 kDa monomer with no symmetry [ 22 ], while lumazine synthase (LS) is a 1.03 MDa 60-mer with icosahedral symmetry [ 23 ]. Density maps of these were calculated from the crystal structures, randomly projected, gaussian noise added to the projections ( Figure 1a ), and the resultant images aligned to the density maps using all information up to Nyquist frequency (2 Å in these cases).…”

Section: Resultsmentioning

confidence: 99%

“…Two structures were selected, the first is the asymmetric proteinase K (PK) ( http://www.rcsb.org , PDB-3DYB [ 22 ]), and the second is the icosahedral lumazine synthase (LS) ( http://www.rcsb.org , PDB-1NQX [ 23 ]). Both were converted to 3D density maps using the program bsf [ 9 ] with a pixel size of 1 Å and sizes of 80 3 and 200 3 , respectively.…”

Section: Methodsmentioning

confidence: 99%

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Validation of 3D EM Reconstructions: The Phantom in the Noise

Heymann

2015

AIMS Biophysics

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Validation is a necessity to trust the structures solved by electron microscopy by single particle techniques. The impressive achievements in single particle reconstruction fuel its expansion beyond a small community of image processing experts. This poses the risk of inappropriate data processing with dubious results. Nowhere is it more clearly illustrated than in the recovery of a reference density map from pure noise aligned to that map—a phantom in the noise. Appropriate use of existing validating methods such as resolution-limited alignment and the processing of independent data sets (“gold standard”) avoid this pitfall. However, these methods can be undermined by biases introduced in various subtle ways. How can we test that a map is a coherent structure present in the images selected from the micrographs? In stead of viewing the phantom emerging from noise as a cautionary tale, it should be used as a defining baseline. Any map is always recoverable from noise images, provided a sufficient number of images are aligned and used in reconstruction. However, with smaller numbers of images, the expected coherence in the real particle images should yield better reconstructions than equivalent numbers of noise or background images, even without masking or imposing resolution limits as potential biases. The validation test proposed is therefore a simple alignment of a limited number of micrograph and noise images against the final reconstruction as reference, demonstrating that the micrograph images yield a better reconstruction. I examine synthetic cases to relate the resolution of a reconstruction to the alignment error as a function of the signal-to-noise ratio. I also administered the test to real cases of publicly available data. Adopting such a test can aid the microscopist in assessing the usefulness of the micrographs taken before committing to lengthy processing with questionable outcomes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Validation of 3D EM Reconstructions: The Phantom in the Noise

Heymann

2015

AIMS Biophysics

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“…McPherson and co-workers have demonstrated that proteins can and will select specific small molecules from a multicomponent solution and that often these compounds can be structurally resolved Larson, Day, Nguyen et al, 2010;Larson et al, 2009aLarson et al, ,b, 2008Larson et al, , 2007b.…”

Section: Discussionmentioning

confidence: 99%

Crystal cookery – using high-throughput technologies and the grocery store as a teaching tool

et al. 2010

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Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience.

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“…P06873). PROK is a study model of serine proteases (Larson et al, 2009). The materials and methods used to obtain PROK crystals, including the soaking and harvesting procedures, are described in Section S1.…”

Section: Figurementioning

confidence: 99%

FragMAXapp: crystallographic fragment-screening data-analysis and project-management system

Lima

Jagudin

Talibov

et al. 2021

Acta Cryst Sect D Struct Biol

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Crystallographic fragment screening (CFS) has become one of the major techniques for screening compounds in the early stages of drug-discovery projects. Following the advances in automation and throughput at modern macromolecular crystallography beamlines, the bottleneck for CFS has shifted from collecting data to organizing and handling the analysis of such projects. The complexity that emerges from the use of multiple methods for processing and refinement and to search for ligands requires an equally sophisticated solution to summarize the output, allowing researchers to focus on the scientific questions instead of on software technicalities. FragMAXapp is the fragment-screening project-management tool designed to handle CFS projects at MAX IV Laboratory. It benefits from the powerful computing infrastructure of large-scale facilities and, as a web application, it is accessible from everywhere.

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High-resolution structure of proteinase K cocrystallized with digalacturonic acid

Cited by 5 publications

References 17 publications

Validation of 3D EM Reconstructions: The Phantom in the Noise

Validation of 3D EM Reconstructions: The Phantom in the Noise

Crystal cookery – using high-throughput technologies and the grocery store as a teaching tool

FragMAXapp: crystallographic fragment-screening data-analysis and project-management system

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