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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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Superposition of NCS-Related ParticlesThe four particles in the ASU were superimposed in O (Jones et al., 1991) using the Cα atoms of Sm protein residues within the Sm fold, U1-70K residues 9–23, and the P atoms of U1 snRNA nucleotides 1–16 and 48–134. Particles B, C, and D were transformed onto particle A with rms of 1.75 Å, 2.03 Å, and 1.97 Å for those Cα and P atoms, respectively. The particles are shown as ribbons with A, B, C, and D in blue, green, yellow, and red, respectively. The substructures that varied in their relative orientations between particles, and which were treated as separate domains in averaging, are indicated. SL1, U1-70K RBD, and U1-70K helix (63–89) were combined and treated as a single domain. SL4 and U1-C C-terminal helix (32–61) were both treated as separate domains.
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fig3: Superposition of NCS-Related ParticlesThe four particles in the ASU were superimposed in O (Jones et al., 1991) using the Cα atoms of Sm protein residues within the Sm fold, U1-70K residues 9–23, and the P atoms of U1 snRNA nucleotides 1–16 and 48–134. Particles B, C, and D were transformed onto particle A with rms of 1.75 Å, 2.03 Å, and 1.97 Å for those Cα and P atoms, respectively. The particles are shown as ribbons with A, B, C, and D in blue, green, yellow, and red, respectively. The substructures that varied in their relative orientations between particles, and which were treated as separate domains in averaging, are indicated. SL1, U1-70K RBD, and U1-70K helix (63–89) were combined and treated as a single domain. SL4 and U1-C C-terminal helix (32–61) were both treated as separate domains.

Mentions: Attempts to superimpose the four U1 snRNP complexes in the ASU (Figure 3) showed that there were small but significant differences between the positions of the following substructures: (1) RNA residues 1–16 and 48–134, U1-C residues 4–31, U1-70K residues 9–23, and proteins Sm-D3, B, D1, D2, F, E, and G; (2) SL1, U1-70K RBD, and residues 63–89; (3) U1-C residues 32–61; (4) SL4. NCS transformation matrices between the substructures in the four U1 snRNP particles within the ASU were generated in O (Jones et al., 1991). Masks were created for each of these substructures using the program NCSMASK (CCP4, 1994). The masks, the solvent-flattened phases at 6.5 Å, the SeMet Sm core data, and the U1-C(Q39C)-EMTS crystal data were used for multidomain, multi-crystal averaging in the program DMMULTI (Cowtan et al., 2001). This resulted in phases with mean figures of merit of 0.623 to 5.5 Å, which compares well with a value of 0.652 to 6.5 Å for the phases used to calculate the experimental map. The resulting 5.5 Å map was clearly of higher quality than the original map: density for β sheets of the Sm proteins became continuous and some RNA density revealed phosphate group bumps. This enabled the U1 snRNP model to be built with more accuracy and certainty than had been possible with the original 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)

Superposition of NCS-Related ParticlesThe four particles in the ASU were superimposed in O (Jones et al., 1991) using the Cα atoms of Sm protein residues within the Sm fold, U1-70K residues 9–23, and the P atoms of U1 snRNA nucleotides 1–16 and 48–134. Particles B, C, and D were transformed onto particle A with rms of 1.75 Å, 2.03 Å, and 1.97 Å for those Cα and P atoms, respectively. The particles are shown as ribbons with A, B, C, and D in blue, green, yellow, and red, respectively. The substructures that varied in their relative orientations between particles, and which were treated as separate domains in averaging, are indicated. SL1, U1-70K RBD, and U1-70K helix (63–89) were combined and treated as a single domain. SL4 and U1-C C-terminal helix (32–61) were both treated as separate domains.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Superposition of NCS-Related ParticlesThe four particles in the ASU were superimposed in O (Jones et al., 1991) using the Cα atoms of Sm protein residues within the Sm fold, U1-70K residues 9–23, and the P atoms of U1 snRNA nucleotides 1–16 and 48–134. Particles B, C, and D were transformed onto particle A with rms of 1.75 Å, 2.03 Å, and 1.97 Å for those Cα and P atoms, respectively. The particles are shown as ribbons with A, B, C, and D in blue, green, yellow, and red, respectively. The substructures that varied in their relative orientations between particles, and which were treated as separate domains in averaging, are indicated. SL1, U1-70K RBD, and U1-70K helix (63–89) were combined and treated as a single domain. SL4 and U1-C C-terminal helix (32–61) were both treated as separate domains.
Mentions: Attempts to superimpose the four U1 snRNP complexes in the ASU (Figure 3) showed that there were small but significant differences between the positions of the following substructures: (1) RNA residues 1–16 and 48–134, U1-C residues 4–31, U1-70K residues 9–23, and proteins Sm-D3, B, D1, D2, F, E, and G; (2) SL1, U1-70K RBD, and residues 63–89; (3) U1-C residues 32–61; (4) SL4. NCS transformation matrices between the substructures in the four U1 snRNP particles within the ASU were generated in O (Jones et al., 1991). Masks were created for each of these substructures using the program NCSMASK (CCP4, 1994). The masks, the solvent-flattened phases at 6.5 Å, the SeMet Sm core data, and the U1-C(Q39C)-EMTS crystal data were used for multidomain, multi-crystal averaging in the program DMMULTI (Cowtan et al., 2001). This resulted in phases with mean figures of merit of 0.623 to 5.5 Å, which compares well with a value of 0.652 to 6.5 Å for the phases used to calculate the experimental map. The resulting 5.5 Å map was clearly of higher quality than the original map: density for β sheets of the Sm proteins became continuous and some RNA density revealed phosphate group bumps. This enabled the U1 snRNP model to be built with more accuracy and certainty than had been possible with the original 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