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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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Positioning of Sm Proteins from U4 Core Structure onto U1 snRNP Selenium Peak PositionsThe anomalous difference map was calculated using data from U1 snRNP crystals that contained SeMet-labeled Sm proteins. The anomalous peaks are contoured at 3σ (red mesh) and 2σ (blue mesh). The selenium anomalous peak positions are shown as red spheres. The Sm protein ring of the U4 core domain is shown as a cartoon with methionine residues colored red.
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fig4: Positioning of Sm Proteins from U4 Core Structure onto U1 snRNP Selenium Peak PositionsThe anomalous difference map was calculated using data from U1 snRNP crystals that contained SeMet-labeled Sm proteins. The anomalous peaks are contoured at 3σ (red mesh) and 2σ (blue mesh). The selenium anomalous peak positions are shown as red spheres. The Sm protein ring of the U4 core domain is shown as a cartoon with methionine residues colored red.

Mentions: We have shown that the use of heavy atom landmarks is a powerful method for the interpretation of low-resolution structure. Since NCS and multi-crystal averaging had improved the quality of the map considerably, we used the improved phases to recalculate anomalous difference maps of the U1 snRNP crystals containing the Se-Met derivative of Sm proteins (Figure 4). The seven Sm proteins contain a total of 25 methionines within the Sm fold, most of which we expected to be ordered in the crystal. We observed 20 anomalous peaks above background (for particle A), which have been assigned to single methionines. All but one (Met11 of SmD2) are within the Sm fold. Furthermore, two of the peaks appear to be composite peaks arising from groups of three (SmB: Met-9, Met-38, and Met-80) and two (SmE: Met-78 and SmF: Met-40) methionines. We assume that the selenium atoms of these residues are close enough in space for the peaks to merge at 6.0 Å resolution. Two of the methionines in this particle have no corresponding selenium signal (SmD1: Met-36 and SmF: Met-27), presumably because the methionine side chains are disordered in our crystals. The assignment of methionines to selenium anomalous peaks is similar for the other three particles. The mean rmsd of the overlays between anomalous peak positions and the methionine sulfur positions in the U4 core domain for the four U1 particles is 2.22 ± 0.08 Å, excluding any composite peaks. The deviations in position arise from several sources: coordinate error of the U1 selenium peaks, coordinate error of the U4 core structure, and genuine differences between the U1 and U4 structures. The latter may arise from the Sm proteins being bound to distinct RNAs, making different crystal contacts and the crystals being grown under different conditions (A.K.W.L., J.L., and K.N., unpublished data). A peak found outside the Sm fold was connected to SmD2 by a kinked rod-like density suggesting α helices. This peak is attributed to Met11 of SmD2. If α helices are built into the density, extending the N-terminal region from that seen in the SmD1D2 heterodimer (Kambach et al., 1999), then Met11 can account for the peak. The U1 map reveals other regions that have shifted relative to the U4 core structure. Many of these are at the N and C termini, outside the canonical Sm fold. One of the most notable changes is for SmF protein residues 6 to 15. There are also conformational differences in some loop regions within the Sm fold, such as SmD3 residues 49 to 55 between strands β3 and β4 and SmF residues 46 to 56, which are explainable by interaction with the N-terminal peptide of U1-70K and with a neighboring complex, respectively. Overall, the structures appear to be the same within the canonical Sm fold.


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

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

Positioning of Sm Proteins from U4 Core Structure onto U1 snRNP Selenium Peak PositionsThe anomalous difference map was calculated using data from U1 snRNP crystals that contained SeMet-labeled Sm proteins. The anomalous peaks are contoured at 3σ (red mesh) and 2σ (blue mesh). The selenium anomalous peak positions are shown as red spheres. The Sm protein ring of the U4 core domain is shown as a cartoon with methionine residues colored red.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Positioning of Sm Proteins from U4 Core Structure onto U1 snRNP Selenium Peak PositionsThe anomalous difference map was calculated using data from U1 snRNP crystals that contained SeMet-labeled Sm proteins. The anomalous peaks are contoured at 3σ (red mesh) and 2σ (blue mesh). The selenium anomalous peak positions are shown as red spheres. The Sm protein ring of the U4 core domain is shown as a cartoon with methionine residues colored red.
Mentions: We have shown that the use of heavy atom landmarks is a powerful method for the interpretation of low-resolution structure. Since NCS and multi-crystal averaging had improved the quality of the map considerably, we used the improved phases to recalculate anomalous difference maps of the U1 snRNP crystals containing the Se-Met derivative of Sm proteins (Figure 4). The seven Sm proteins contain a total of 25 methionines within the Sm fold, most of which we expected to be ordered in the crystal. We observed 20 anomalous peaks above background (for particle A), which have been assigned to single methionines. All but one (Met11 of SmD2) are within the Sm fold. Furthermore, two of the peaks appear to be composite peaks arising from groups of three (SmB: Met-9, Met-38, and Met-80) and two (SmE: Met-78 and SmF: Met-40) methionines. We assume that the selenium atoms of these residues are close enough in space for the peaks to merge at 6.0 Å resolution. Two of the methionines in this particle have no corresponding selenium signal (SmD1: Met-36 and SmF: Met-27), presumably because the methionine side chains are disordered in our crystals. The assignment of methionines to selenium anomalous peaks is similar for the other three particles. The mean rmsd of the overlays between anomalous peak positions and the methionine sulfur positions in the U4 core domain for the four U1 particles is 2.22 ± 0.08 Å, excluding any composite peaks. The deviations in position arise from several sources: coordinate error of the U1 selenium peaks, coordinate error of the U4 core structure, and genuine differences between the U1 and U4 structures. The latter may arise from the Sm proteins being bound to distinct RNAs, making different crystal contacts and the crystals being grown under different conditions (A.K.W.L., J.L., and K.N., unpublished data). A peak found outside the Sm fold was connected to SmD2 by a kinked rod-like density suggesting α helices. This peak is attributed to Met11 of SmD2. If α helices are built into the density, extending the N-terminal region from that seen in the SmD1D2 heterodimer (Kambach et al., 1999), then Met11 can account for the peak. The U1 map reveals other regions that have shifted relative to the U4 core structure. Many of these are at the N and C termini, outside the canonical Sm fold. One of the most notable changes is for SmF protein residues 6 to 15. There are also conformational differences in some loop regions within the Sm fold, such as SmD3 residues 49 to 55 between strands β3 and β4 and SmF residues 46 to 56, which are explainable by interaction with the N-terminal peptide of U1-70K and with a neighboring complex, respectively. Overall, the structures appear to be the same within the canonical Sm fold.

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