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Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.

Oubridge C, Krummel DA, Leung AK, Li J, Nagai K - Structure (2009)

Bottom Line: We were able to locate anomalous scatterers with positional errors below 2 A.This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence.This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, England, UK.

ABSTRACT
We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

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Related in: MedlinePlus

Locating Ta6Br12 Clusters(A) Three z sections of an anomalous Patterson map calculated from the inflection data of a two wavelength anomalous dispersion experiment. Cross-peaks for all four major sites (origin, 1-2, 1-3, and 1-4) and for one minor site (1-6) can be seen on these sections, as well as a number of other cross-peaks.(B) The major and minor Ta6Br12 binding sites within the ASU are respectively indicated by large and small magenta spheres and numbered. The four U1 snRNPs in the ASU (particles A, B, C, and D) are colored red, yellow, green, and blue, respectively, with protein shown as ribbon and RNA as cartoon. The Ta6Br12 clusters bind in cavities between protein and RNA. Major sites lie between SL4 nucleotides 138 and 139 of RNA and the Sm ring residues in the β1-β2 loop of SmB and the β3-β4 loop of SmD1. Minor sites are found between the long α helix of U1-C protein, near Trp41, and the base of SL3 of a NCS-related particle.
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fig1: Locating Ta6Br12 Clusters(A) Three z sections of an anomalous Patterson map calculated from the inflection data of a two wavelength anomalous dispersion experiment. Cross-peaks for all four major sites (origin, 1-2, 1-3, and 1-4) and for one minor site (1-6) can be seen on these sections, as well as a number of other cross-peaks.(B) The major and minor Ta6Br12 binding sites within the ASU are respectively indicated by large and small magenta spheres and numbered. The four U1 snRNPs in the ASU (particles A, B, C, and D) are colored red, yellow, green, and blue, respectively, with protein shown as ribbon and RNA as cartoon. The Ta6Br12 clusters bind in cavities between protein and RNA. Major sites lie between SL4 nucleotides 138 and 139 of RNA and the Sm ring residues in the β1-β2 loop of SmB and the β3-β4 loop of SmD1. Minor sites are found between the long α helix of U1-C protein, near Trp41, and the base of SL3 of a NCS-related particle.

Mentions: A multiwavelength anomalous dispersion data set was collected from a tantalum bromide cluster (Ta6Br12) derivative (Knäblein et al., 1997) at the Ta L-III edge at two wavelengths: inflection (1.2557 Å) and remote (1.2511 Å). The inflection data were used to calculate an anomalous Patterson map (Figure 1A) and the coordinates of four Ta6Br12 sites were obtained manually from the cross-peaks. Ta6Br12 cluster coordinates and occupancies were refined in SHARP (de la Fortelle and Bricogne, 1997). Inspection of residual maps showed four additional minor sites with lower occupancy. Each minor site was 48 Å from a major site, confirming that there were four U1 snRNPs in the ASU, related by noncrystallographic symmetry (NCS), and each bound to two Ta6Br12 clusters. Spherically averaged form factors of the clusters at 7 Å resolution resulted in higher final phasing power (1.51 versus 1.25), lower Cullis R factor (0.71 versus 0.76), and better overall figures of merit (0.413 versus 0.404) than a single point Gaussian model. Figure 1B shows the packing of four U1 snRNPs in the unit cell and the positions of the four major and four minor Ta sites. The sites were refined with and without coordinate inversion, and the phases were subjected to solvent flipping in Solomon (Abrahams and Leslie, 1996) with a 60% solvent content and extended from 7.5 to 7.0 Å over 11 cycles. The correct hand was identified from better figures of merit for the solvent flattened phases (0.541 versus 0.531) and clear density for A-form RNA in the resulting electron density map.


Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.

Oubridge C, Krummel DA, Leung AK, Li J, Nagai K - Structure (2009)

Locating Ta6Br12 Clusters(A) Three z sections of an anomalous Patterson map calculated from the inflection data of a two wavelength anomalous dispersion experiment. Cross-peaks for all four major sites (origin, 1-2, 1-3, and 1-4) and for one minor site (1-6) can be seen on these sections, as well as a number of other cross-peaks.(B) The major and minor Ta6Br12 binding sites within the ASU are respectively indicated by large and small magenta spheres and numbered. The four U1 snRNPs in the ASU (particles A, B, C, and D) are colored red, yellow, green, and blue, respectively, with protein shown as ribbon and RNA as cartoon. The Ta6Br12 clusters bind in cavities between protein and RNA. Major sites lie between SL4 nucleotides 138 and 139 of RNA and the Sm ring residues in the β1-β2 loop of SmB and the β3-β4 loop of SmD1. Minor sites are found between the long α helix of U1-C protein, near Trp41, and the base of SL3 of a NCS-related particle.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712673&req=5

fig1: Locating Ta6Br12 Clusters(A) Three z sections of an anomalous Patterson map calculated from the inflection data of a two wavelength anomalous dispersion experiment. Cross-peaks for all four major sites (origin, 1-2, 1-3, and 1-4) and for one minor site (1-6) can be seen on these sections, as well as a number of other cross-peaks.(B) The major and minor Ta6Br12 binding sites within the ASU are respectively indicated by large and small magenta spheres and numbered. The four U1 snRNPs in the ASU (particles A, B, C, and D) are colored red, yellow, green, and blue, respectively, with protein shown as ribbon and RNA as cartoon. The Ta6Br12 clusters bind in cavities between protein and RNA. Major sites lie between SL4 nucleotides 138 and 139 of RNA and the Sm ring residues in the β1-β2 loop of SmB and the β3-β4 loop of SmD1. Minor sites are found between the long α helix of U1-C protein, near Trp41, and the base of SL3 of a NCS-related particle.
Mentions: A multiwavelength anomalous dispersion data set was collected from a tantalum bromide cluster (Ta6Br12) derivative (Knäblein et al., 1997) at the Ta L-III edge at two wavelengths: inflection (1.2557 Å) and remote (1.2511 Å). The inflection data were used to calculate an anomalous Patterson map (Figure 1A) and the coordinates of four Ta6Br12 sites were obtained manually from the cross-peaks. Ta6Br12 cluster coordinates and occupancies were refined in SHARP (de la Fortelle and Bricogne, 1997). Inspection of residual maps showed four additional minor sites with lower occupancy. Each minor site was 48 Å from a major site, confirming that there were four U1 snRNPs in the ASU, related by noncrystallographic symmetry (NCS), and each bound to two Ta6Br12 clusters. Spherically averaged form factors of the clusters at 7 Å resolution resulted in higher final phasing power (1.51 versus 1.25), lower Cullis R factor (0.71 versus 0.76), and better overall figures of merit (0.413 versus 0.404) than a single point Gaussian model. Figure 1B shows the packing of four U1 snRNPs in the unit cell and the positions of the four major and four minor Ta sites. The sites were refined with and without coordinate inversion, and the phases were subjected to solvent flipping in Solomon (Abrahams and Leslie, 1996) with a 60% solvent content and extended from 7.5 to 7.0 Å over 11 cycles. The correct hand was identified from better figures of merit for the solvent flattened phases (0.541 versus 0.531) and clear density for A-form RNA in the resulting electron density map.

Bottom Line: We were able to locate anomalous scatterers with positional errors below 2 A.This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence.This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, England, UK.

ABSTRACT
We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Show MeSH
Related in: MedlinePlus