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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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Overlay of Selenium Peaks from Multiple Crystals Containing U1-70K SeMet Mutant Protein(A) U1-70K residues 61–180 are shown as orange cartoon, with part of U1 snRNA, including SL1, shown in light gray. The selenium peak coordinates from anomalous maps of the eight U1-70K mutants are marked by colored spheres. The selenium anomalous maps are shown, all contoured at 3.5 σ and colored to match the spheres. Sphere diameter is ∼2 Å. As well as the four natural methionines (67, 88, 134, and 157), which have corresponding peaks in all the mutants, two of the mutant site peaks (E61M and I75M) are also shown. The colors are: wild-type, black; L9M, red; I19M, orange; E31M, light green; I41M, dark green; I49M, cyan; E61M, blue; I75M, dark blue.(B) The path of the extended N terminus of U1-70K. Electron density attributed to U1-70K is shown in brown and contoured at 1 σ. Where density is absent, approximately between residues 24 and 45, a plausible path for the peptide is indicated based on the selenium positions of E31M and I41M. Selenium peaks and anomalous maps are as for (A). Near the selenium site of L9M, U1-70K is seen to interact with U1-C, which is red.
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fig2: Overlay of Selenium Peaks from Multiple Crystals Containing U1-70K SeMet Mutant Protein(A) U1-70K residues 61–180 are shown as orange cartoon, with part of U1 snRNA, including SL1, shown in light gray. The selenium peak coordinates from anomalous maps of the eight U1-70K mutants are marked by colored spheres. The selenium anomalous maps are shown, all contoured at 3.5 σ and colored to match the spheres. Sphere diameter is ∼2 Å. As well as the four natural methionines (67, 88, 134, and 157), which have corresponding peaks in all the mutants, two of the mutant site peaks (E61M and I75M) are also shown. The colors are: wild-type, black; L9M, red; I19M, orange; E31M, light green; I41M, dark green; I49M, cyan; E61M, blue; I75M, dark blue.(B) The path of the extended N terminus of U1-70K. Electron density attributed to U1-70K is shown in brown and contoured at 1 σ. Where density is absent, approximately between residues 24 and 45, a plausible path for the peptide is indicated based on the selenium positions of E31M and I41M. Selenium peaks and anomalous maps are as for (A). Near the selenium site of L9M, U1-70K is seen to interact with U1-C, which is red.

Mentions: The RNA binding domain (RBD) of U1-70K was known to bind to SL1 (Patton and Pederson, 1988; Query et al., 1989) and a large globule of electron density was seen in the SL1 loop region. The initial map did not permit unambiguous fitting of the RBD, so U1-70K was labeled with SeMet and reconstituted into U1 snRNP. The resulting crystal gave four Se anomalous peaks (above 4.0 σ) per U1 particle. Two of the peaks, corresponding to Met-134 and Met-157, lie within the RBD (Figure 2A). The RBD was homology modeled from the N-terminal RBD of U1A (Nagai et al., 1990; Oubridge et al., 1994) and placed using these peaks along with the rod-like density of its two α helices. The loop region of SL1 was built in such a way that G28 and U30 are in close proximities of Tyr112 and Leu175, which were assumed from cross-linking data (Urlaub et al., 2000). The remaining two anomalous peaks were found in the long rod-like density adjacent to SL1. This region was predicted to be α-helical and was modeled as such between residues 63 and 89. Further support for this model comes from the observation that many of the basic residues of the helix are close to the phosphate backbone of SL1, favoring electrostatic interactions (Pomeranz Krummel et al., 2009).


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

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

Overlay of Selenium Peaks from Multiple Crystals Containing U1-70K SeMet Mutant Protein(A) U1-70K residues 61–180 are shown as orange cartoon, with part of U1 snRNA, including SL1, shown in light gray. The selenium peak coordinates from anomalous maps of the eight U1-70K mutants are marked by colored spheres. The selenium anomalous maps are shown, all contoured at 3.5 σ and colored to match the spheres. Sphere diameter is ∼2 Å. As well as the four natural methionines (67, 88, 134, and 157), which have corresponding peaks in all the mutants, two of the mutant site peaks (E61M and I75M) are also shown. The colors are: wild-type, black; L9M, red; I19M, orange; E31M, light green; I41M, dark green; I49M, cyan; E61M, blue; I75M, dark blue.(B) The path of the extended N terminus of U1-70K. Electron density attributed to U1-70K is shown in brown and contoured at 1 σ. Where density is absent, approximately between residues 24 and 45, a plausible path for the peptide is indicated based on the selenium positions of E31M and I41M. Selenium peaks and anomalous maps are as for (A). Near the selenium site of L9M, U1-70K is seen to interact with U1-C, which is red.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Overlay of Selenium Peaks from Multiple Crystals Containing U1-70K SeMet Mutant Protein(A) U1-70K residues 61–180 are shown as orange cartoon, with part of U1 snRNA, including SL1, shown in light gray. The selenium peak coordinates from anomalous maps of the eight U1-70K mutants are marked by colored spheres. The selenium anomalous maps are shown, all contoured at 3.5 σ and colored to match the spheres. Sphere diameter is ∼2 Å. As well as the four natural methionines (67, 88, 134, and 157), which have corresponding peaks in all the mutants, two of the mutant site peaks (E61M and I75M) are also shown. The colors are: wild-type, black; L9M, red; I19M, orange; E31M, light green; I41M, dark green; I49M, cyan; E61M, blue; I75M, dark blue.(B) The path of the extended N terminus of U1-70K. Electron density attributed to U1-70K is shown in brown and contoured at 1 σ. Where density is absent, approximately between residues 24 and 45, a plausible path for the peptide is indicated based on the selenium positions of E31M and I41M. Selenium peaks and anomalous maps are as for (A). Near the selenium site of L9M, U1-70K is seen to interact with U1-C, which is red.
Mentions: The RNA binding domain (RBD) of U1-70K was known to bind to SL1 (Patton and Pederson, 1988; Query et al., 1989) and a large globule of electron density was seen in the SL1 loop region. The initial map did not permit unambiguous fitting of the RBD, so U1-70K was labeled with SeMet and reconstituted into U1 snRNP. The resulting crystal gave four Se anomalous peaks (above 4.0 σ) per U1 particle. Two of the peaks, corresponding to Met-134 and Met-157, lie within the RBD (Figure 2A). The RBD was homology modeled from the N-terminal RBD of U1A (Nagai et al., 1990; Oubridge et al., 1994) and placed using these peaks along with the rod-like density of its two α helices. The loop region of SL1 was built in such a way that G28 and U30 are in close proximities of Tyr112 and Leu175, which were assumed from cross-linking data (Urlaub et al., 2000). The remaining two anomalous peaks were found in the long rod-like density adjacent to SL1. This region was predicted to be α-helical and was modeled as such between residues 63 and 89. Further support for this model comes from the observation that many of the basic residues of the helix are close to the phosphate backbone of SL1, favoring electrostatic interactions (Pomeranz Krummel et al., 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.

Show MeSH