Bias in cross-validated free<i>R</i>factors: mitigation of the effects of non-crystallographic symmetry

Fabiola, Felcy; Коростелев, А.A.; Chapman, Michael S.

doi:10.1107/s0907444905040680

Cited by 29 publications

(30 citation statements)

References 26 publications

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“…The structure factors of our free R test set were randomly selected before the model was built. Because of the 6-fold NCS in the asymmetric unit the test set for the free R is not completely independent of the working R set (41). Calculating a truly independent free R would be challenging and would require us to start over from the diffraction data.…”

Section: Methodsmentioning

confidence: 99%

The structure of LepA, the ribosomal back translocase

Evans¹,

Blaha²,

Bailey

et al. 2008

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

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LepA is a highly conserved elongation factor that promotes the back translocation of tRNAs on the ribosome during the elongation cycle. We have determined the crystal structure of LepA from Escherichia coli at 2.8-Å resolution. The high degree of sequence identity between LepA and EF-G is reflected in the structural similarity between the individual homologous domains of LepA and EF-G. However, the orientation of domains III and V in LepA differs from their orientations in EF-G. LepA also contains a C-terminal domain (CTD) not found in EF-G that has a previously unobserved protein fold. The high structural similarity between LepA and EF-G enabled us to derive a homology model for LepA bound to the ribosome using a 7.3-Å cryo-EM structure of a complex between EF-G and the 70S ribosome. In this model, the very electrostatically positive CTD of LepA is placed in the direct vicinity of the A site of the large ribosomal subunit, suggesting a possible interaction between the CTD and the back translocated tRNA or 23S rRNA.back translocation ͉ crystal structure ͉ ribosome ͉ elongation factor

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Section: Methodsmentioning

confidence: 99%

The structure of LepA, the ribosomal back translocase

Evans¹,

Blaha²,

Bailey

et al. 2008

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

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“…Real space refinement was carried out manually using the Coot graphics program (15), followed by positional B-factor refinement in the Crystallography & NMR System (CNS) (9). Cross-validation is not reported, since the test reflections are related to working reflections by the 60-fold noncrystallographic symmetry (16).…”

Section: Fig 1 Calcium-binding Sites In Flock House Virus (A)mentioning

confidence: 99%

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

Banerjee

Speir

Kwan³

et al. 2010

J Virol

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Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membranedisrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.During cellular entry, the capsids of nonenveloped viruses undergo conformational changes triggered by various host factors. The transitions include the exposure of membrane-active, hydrophobic viral polypeptides and the destabilization and disassembly of the capsid, culminating in the delivery of the viral genome to the cytoplasm. A detailed, stepwise pathway of disassembly is unavailable for most viruses and requires molecular characterization of the intermediates formed during this process. Replication of disassembly intermediates in vitro necessitates careful treatment of native virions to simulate conditions likely to be encountered inside host cells. Receptor binding induces conformational changes in poliovirus, and the poliovirus entry intermediates, the 135S and 80S particles, can be generated in vitro by treating the native virion with the purified receptor, or by heating (6,10,19). Reovirus, which requires cathepsin-mediated proteolysis in the late endosomes in order to undergo entry-related changes, produces infectious subvirion particles (ISVP) and core particles as disassembly intermediates upon treatment with proteases in vitro (11,14).Apart from receptors and cellular proteases, other host determinants affecting viruses include low pH and Ca 2ϩ depletion in specialized cellular compartments. The removal of Ca 2ϩ from capsids could be particularly crucial for viruses that depend on metal ions for assembly and stability, such as rotavirus, mouse polyomavirus, and dragon grouper nervous necrosis virus (DGNNV) (1,39,43). A few studies have reported that mutations at the metal-binding sites decrease virus infectivity by impairing the early stages of virus-host interaction and genome release (23,24). When Ca 2ϩ is removed from the capsids of plant viruses such as cowpea chlorotic mottle virus (CCMV), tomato bushy stunt virus (TBSV), and turnip crinkle virus (TCV) by metal chelators (2, 33, 35), the virion swells by almost 10% and becomes suscept...

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“…On the other, there are a number of hypothetical reasons that could decrease the reliability of R free statistics for low resolutions. For example, a smaller number of reflections may make test sets and corresponding statistics poorer, reflections from the test sets may be indirectly related to those from the work sets for structures with local symmetries (Fabiola et al, 2006; as discussed in x3.2, such structures are more frequent at lower resolutions) etc.…”

Section: R Free and Difference R Free à Rmentioning

confidence: 99%

On the use of logarithmic scales for analysis of diffraction data

Urzhumtsev

Afonine

Adams

2009

Acta Crystallogr D Biol Cryst

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Predictions of the possible model parameterization and of the values of model characteristics such as R factors are important for macromolecular refinement and validation protocols. One of the key parameters defining these and other values is the resolution of the experimentally measured diffraction data. The higher the resolution, the larger the number of diffraction data N ref , the larger its ratio to the number N at of non-H atoms, the more parameters per atom can be used for modelling and the more precise and detailed a model can be obtained. The ratio N ref /N at was calculated for models deposited in the Protein Data Bank as a function of the resolution at which the structures were reported. The most frequent values for this distribution depend essentially linearly on resolution when the latter is expressed on a uniform logarithmic scale. This defines simple analytic formulae for the typical Matthews coefficient and for the typically allowed number of parameters per atom for crystals diffracting to a given resolution. This simple dependence makes it possible in many cases to estimate the expected resolution of the experimental data for a crystal with a given Matthews coefficient. When expressed using the same logarithmic scale, the most frequent values for R and R free factors and for their difference are also essentially linear across a large resolution range. The minimal R-factor values are practically constant at resolutions better than 3 Å , below which they begin to grow sharply. This simple dependence on the resolution allows the prediction of expected R-factor values for unknown structures and may be used to guide model refinement and validation.

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Bias in cross-validated freeRfactors: mitigation of the effects of non-crystallographic symmetry

Cited by 29 publications

References 26 publications

The structure of LepA, the ribosomal back translocase

The structure of LepA, the ribosomal back translocase

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

On the use of logarithmic scales for analysis of diffraction data

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