FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2021506799> ?p ?o ?g. }

• W2021506799 endingPage "938" @default.
• W2021506799 startingPage "930" @default.
• W2021506799 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 Å resolution electron density map. At 5–7 Å resolution, α helices and β 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 Å. 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. 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 Å resolution electron density map. At 5–7 Å resolution, α helices and β 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 Å. 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. IntroductionMost proteins in eukaryotic cells exist as components of large protein, RNA-protein, or DNA-protein complexes, which carry out important biological functions in an integrated manner (Gavin et al., 2002Gavin A.C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. et al.Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (3972) Google Scholar). The structure of individual components of these complexes often provides little insight into the structure and mechanism of the assembly of which they are a part. Therefore, the structure of all, or a major part of the assembly must be determined. For example, the structure of individual ribosomal proteins provided little or no insight into protein synthesis or decoding (Ramakrishnan and White, 1998Ramakrishnan V. White S.W. Ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome.Trends Biochem. Sci. 1998; 23: 208-212Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Therefore, to understand higher order functions of the cell it is important to undertake crystallographic studies of large macromolecular assemblies (Ramakrishnan, 2002Ramakrishnan V. Ribosome structure and the mechanism of translation.Cell. 2002; 108: 557-572Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, Klein et al., 2004Klein D.J. Moore P.B. Steitz T.A. The roles of ribosomal proteins in the structure, assembly and evolution of the large ribosomal subunit.J. Mol. Biol. 2004; 340: 141-177Crossref PubMed Scopus (344) Google Scholar). However, it is very difficult to purify many of these assemblies from natural sources or to assemble them from recombinant components in quantities sufficient for crystallographic studies (Maier et al., 2006Maier T. Jenni S. Ban N. Architecture of mammalian fatty acid synthase at 4.5 Å resolution.Science. 2006; 311: 1258-1262Crossref PubMed Scopus (301) Google Scholar, Liu et al., 2006Liu Q. Greimann J.C. Lima C.D. Reconstitution, activities, and structure of the eukaryotic RNA exosome.Cell. 2006; 127: 1223-1237Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Crystallization is particularly challenging for large complexes because of sample heterogeneity, which may arise from modification of the sample in vivo, and dissociation of any weakly associated components of the complex as observed with RNA polymerase II (Cramer et al., 2000Cramer P. Bushnell D.A. Fu J. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Architecture of RNA polymerase II and implications for the transcription mechanism.Science. 2000; 288: 640-649Crossref PubMed Scopus (463) Google Scholar) and the ribosome (Wimberly et al., 2000Wimberly B.T. Brodersen D.E. Clemons Jr., W.M. Morgan-Warren R.J. Carter A.P. Vonrhein C. Hartsch T. Ramakrishnan V. Structure of the 30S ribosomal subunit.Nature. 2000; 407: 327-339Crossref PubMed Scopus (1700) Google Scholar). Furthermore, even when these difficulties are overcome, crystals of large complexes often diffract weakly to low resolution and are very susceptible to radiation damage. Fortunately, even low resolution diffraction data (5–7 Å) can provide essential insights into the functions of large macromolecular assemblies, provided one can obtain and interpret an electron density map (Ban et al., 1999Ban N. Nissen P. Hansen J. Capel M. Moore P.B. Steitz T.A. Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit.Nature. 1999; 400: 841-847Crossref PubMed Scopus (348) Google Scholar, Clemons et al., 1999Clemons Jr., W.M. May J.L. Wimberly B.T. McCutcheon J.P. Capel M.S. Ramakrishnan V. Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution.Nature. 1999; 400: 833-840Crossref PubMed Scopus (313) Google Scholar, Murakami et al., 2002Murakami K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex.Science. 2002; 296: 1285-1290Crossref PubMed Scopus (533) Google Scholar, Bushnell et al., 2004Bushnell D.A. Westover K.D. Davis R.E. Kornberg R.D. Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms.Science. 2004; 303: 983-988Crossref PubMed Scopus (270) Google Scholar). At such resolutions, α helices appear as tubular density (Muirhead and Perutz, 1963Muirhead H. Perutz M.F. Structure of haemoglobin. A three-dimensional Fourier synthesis of reduced human haemoglobin at 5.5 Å resolution.Nature. 1963; 199: 633-638Crossref PubMed Scopus (151) Google Scholar) and β sheets as flat density (Ban et al., 1999Ban N. Nissen P. Hansen J. Capel M. Moore P.B. Steitz T.A. Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit.Nature. 1999; 400: 841-847Crossref PubMed Scopus (348) Google Scholar, Clemons et al., 1999Clemons Jr., W.M. May J.L. Wimberly B.T. McCutcheon J.P. Capel M.S. Ramakrishnan V. Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution.Nature. 1999; 400: 833-840Crossref PubMed Scopus (313) Google Scholar). It may be possible to place a protein of known structure into the electron density map at this resolution, particularly if its secondary structure is largely α-helical; it is, however, difficult to follow the polypeptide chain of a protein of unknown structure and determine its fold de novo.We have recently solved the structure, at 5.5 Å resolution, of the functional core of human U1 snRNP, which consists of U1 snRNA, seven Sm proteins, U1-70K, and U1-C (Pomeranz Krummel et al., 2009Pomeranz Krummel D.A. Oubridge C. Leung A.K.W. Li J. Nagai K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution.Nature. 2009; 458: 475-480Crossref PubMed Scopus (243) Google Scholar). In this paper, we show that anomalous peaks from selenium and other heavy atoms could be obtained from highly radiation-sensitive crystals that typically diffracted to ∼6.5 Å. The anomalous peaks were an important aid to fitting known protein folds into the electron density and provided evidence of the quality of the fit. A polypeptide chain of unknown structure was traced on the basis of selenium positions when methionines, which could subsequently be replaced by selenomethionine (SeMet), were introduced by mutagenesis into the sequence at short intervals.ResultsCalculation of an Experimental Electron Density Map at 6.5 Å ResolutionThe reconstitution and crystallization of U1 snRNP have been described (Muto et al., 2004Muto Y. Pomeranz Krummel D. Oubridge C. Hernandez H. Robinson C.V. Neuhaus D. Nagai K. The structure and biochemical properties of the human spliceosomal protein U1C.J. Mol. Biol. 2004; 341: 185-198Crossref PubMed Scopus (44) Google Scholar, Pomeranz Krummel et al., 2009Pomeranz Krummel D.A. Oubridge C. Leung A.K.W. Li J. Nagai K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution.Nature. 2009; 458: 475-480Crossref PubMed Scopus (243) Google Scholar). The functional core of human U1 snRNP consists of U1 snRNA and nine proteins. The crystals grow in P1 space group with unit cell parameters a = 127 Å, b = 128 Å, c = 156 Å, α = 96°, β = 107°, and γ = 101° and diffract to ∼6 Å resolution. Self-rotation and self-Patterson analyses suggested four U1 snRNPs in the asymmetric unit (ASU) (data not shown).A multiwavelength anomalous dispersion data set was collected from a tantalum bromide cluster (Ta6Br12) derivative (Knäblein et al., 1997Knäblein J. Neuefeind T. Schneider F. Bergner A. Messerschmidt A. Löwe J. Steipe B. Huber R. Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography.J. Mol. Biol. 1997; 270: 1-7Crossref PubMed Scopus (50) Google Scholar) 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, 1997de la Fortelle E. Bricogne G. Maximum likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods.in: Carter C.W. Sweet R.M. Methods in Enzymology. Academic Press, New York1997: 472-493Google Scholar). 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, 1996Abrahams J.P. Leslie A.G. Methods used in the structure determination of bovine mitochondrial F1 ATPase.Acta Crystallogr. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1140) Google Scholar) 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.To improve phasing and to aid location of the Sm proteins, they were all labeled with SeMet then incorporated into U1 snRNP crystals. Se K-edge (λ = 0.9797 Å) data were collected and an anomalous difference map calculated using solvent-flattened Ta6Br12 phases. The map had 123 peaks greater than 3.0 SD (σ) above background (data not shown).To further improve phasing and to locate the Zn-finger protein U1-C, an anomalous map was calculated from native data collected at the zinc K edge (λ = 1.2827 Å). The map showed four peaks greater than 7.8 σ with the next strongest being 4.4 σ. The four strongest peaks correspond to zinc atoms in U1-C protein, in agreement with there being four U1 snRNP complexes in the ASU.The coordinates of Se atoms, Zn atoms, and Ta6Br12 clusters were refined together against their respective data sets in SHARP (de la Fortelle and Bricogne, 1997de la Fortelle E. Bricogne G. Maximum likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods.in: Carter C.W. Sweet R.M. Methods in Enzymology. Academic Press, New York1997: 472-493Google Scholar). Occupancy and position were refined for all sites, except for one Ta6Br12 site, which was fixed at the origin. B factors were fixed at 200.0 Å2. The phases were improved by solvent flipping in Solomon with a 60% solvent content, extended from 7.0 Å to 6.5 Å over 11 cycles and had final overall figures of merit of 0.652.Model BuildingRNAThe resulting electron density map was of high quality such that RNA helices, with shallow minor grooves and deep major grooves, were readily discernible. It was not always possible to fit long fragments of A-form RNA into extended regions of helical density, they were therefore initially built from short fragments of idealized A-form RNA and manually rebuilt around the junctions to improve stereochemistry. An NMR structure of a kissing loop RNA (Kim and Tinoco, 2000Kim C.H. Tinoco Jr., I. A retroviral RNA kissing complex containing only two G-C base pairs.Proc. Natl. Acad. Sci. USA. 2000; 97: 9396-9401Crossref PubMed Scopus (97) Google Scholar; PDB code 1f5u) was built into the electron density at the apical part of stem loop 2 (SL2). This domain had been introduced into the RNA to promote crystal lattice interactions (Pomeranz Krummel et al., 2009Pomeranz Krummel D.A. Oubridge C. Leung A.K.W. Li J. Nagai K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution.Nature. 2009; 458: 475-480Crossref PubMed Scopus (243) Google Scholar), which are seen within the ASU between U1 snRNP complexes A and B and complexes C and D (Figure 1B).Sm ProteinsThe structure of the U4 snRNP core domain, an assembly of seven Sm proteins and a 68 nucleotide RNA, was solved to 3.6 Å resolution independently (A.K.W.L., J.L., and K.N., unpublished data) and four of the protein structures have been determined as heterodimers of SmD1D2 and SmBD3 (Kambach et al., 1999Kambach C. Walke S. Young R. Avis J.M. de la Fortelle E. Raker V.A. Lührmann R. Li J. Nagai K. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs.Cell. 1999; 96: 375-387Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). One to one correspondence was found between the majority of our selenium anomalous peaks from SeMet-labeled Sm proteins and the methionine residues in the U4 core domain whereas some anomalous peaks are composite peaks arising from two or more selenium atoms. The U4 core domain structure (A.K.W.L., J.L., and K.N., unpublished data) was initially fitted into the U1 snRNP map by superposition of the U4 core methionine sulfur positions onto the corresponding refined Se sites. In the U1 snRNP map the N-terminal helices of the Sm fold were fitted into tubular densities but the electron density for the β sheet in some subunits was discontinuous.U1-CThe lowest energy NMR structure of the Zn-finger domain of U1-C (Muto et al., 2004Muto Y. Pomeranz Krummel D. Oubridge C. Hernandez H. Robinson C.V. Neuhaus D. Nagai K. The structure and biochemical properties of the human spliceosomal protein U1C.J. Mol. Biol. 2004; 341: 185-198Crossref PubMed Scopus (44) Google Scholar; PDB code 1uw2) was placed at the Zn anomalous peak positions based on the rod-like density of the α-helical region. A long section of α-helical density from U1 snRNP complex A extends toward SL3 of complex C and vice versa, and complexes B and D show the same relationship. This long α-helix of U1-C shows that helices B and C, which form a turn with a flexible loop in isolated U1-C in the solution structure (Muto et al., 2004Muto Y. Pomeranz Krummel D. Oubridge C. Hernandez H. Robinson C.V. Neuhaus D. Nagai K. The structure and biochemical properties of the human spliceosomal protein U1C.J. Mol. Biol. 2004; 341: 185-198Crossref PubMed Scopus (44) Google Scholar), form a continuous helix in the U1 snRNP crystal. In order to ensure correct orientation of the Zn-finger domain a native crystal was treated with ethyl mercury thiosalicylate (EMTS). An anomalous peak of mercury (Hg) bound to Cys-25 adjacent to Zn-coordinating His-24 was used to place the Zn-finger domain more precisely. An additional Hg anomalous peak from a single Cys mutant (Q39C) was used to orient the C-terminal helix and place it in the correct register.U1-70KThe RNA binding domain (RBD) of U1-70K was known to bind to SL1 (Patton and Pederson, 1988Patton J.R. Pederson T. The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5′ stem-loop of U1 RNA and interacts with Sm domain proteins.Proc. Natl. Acad. Sci. USA. 1988; 85: 747-751Crossref PubMed Scopus (40) Google Scholar, Query et al., 1989Query C.C. Bentley R.C. Keene J.D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70k U1 snRNP protein.Cell. 1989; 57: 89-101Abstract Full Text PDF PubMed Scopus (441) Google Scholar) 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., 1990Nagai K. Oubridge C. Jessen T.-H. Li J. Evans P.R. The crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A.Nature. 1990; 348: 515-520Crossref PubMed Scopus (550) Google Scholar, Oubridge et al., 1994Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin.Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar) 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., 2000Urlaub H. Hartmuth K. Kostka S. Grelle G. Lührmann R. A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs.J. Biol. Chem. 2000; 275: 41458-41468Crossref PubMed Scopus (59) Google Scholar). 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., 2009Pomeranz Krummel D.A. Oubridge C. Leung A.K.W. Li J. Nagai K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution.Nature. 2009; 458: 475-480Crossref PubMed Scopus (243) Google Scholar).Figure 2Overlay of Selenium Peaks from Multiple Crystals Containing U1-70K SeMet Mutant ProteinShow full caption(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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The location and the structure of the N-terminal 62 residues of U1-70K were unknown, so we made several single methionine mutants in this region to locate these residues from the position of anomalous peaks when U1 snRNP was reconstituted with their SeMet derivative. The amino acids chosen for substitution with methionine had long and/or hydrophobic side chains, in order to minimize structural changes. Basic side chains were avoided because their mutation could disrupt RNA binding. Substitutions were made at approximately ten residue intervals to facilitate chain tracing. Data from crystals grown with SeMet derivatives of these mutants gave selenium anomalous difference peaks that revealed an extended polypeptide from residue 9 to 63 (Figure 2B). The polypeptide interacts with U1-C protein and traces a path around the Sm protein ring to the beginning of the α helix adjacent to SL1. Figure 2A shows an overlay of Se peaks for the natural methionine residues of U1-70K that are present in all these crystals. The selenium anomalous peaks of these residues are clustered with rms < 1.9 Å, except for Met67 in complex B (rms = 2.5 Å). The observed scatter was within the expected positional errors at this resolution.Phase Extension to 5.5 Å: Multi-NCS, Multi-Crystal AveragingAn EMTS-soaked crystal of U1 snRNP, containing the Q39C mutant of U1-C, diffracted to 5.5 Å, compared with 6.5 Å for the Sm protein SeMet derivative crystal from which, along with the Ta6Br12 derivative and zinc edge data, the original experimental map was calculated. The unit cell of this crystal had a c axis 3.7 Å shorter than the mean of the other crystals' c (152.0 Å compared to average of 155.7 ± 0.7 Å; see Table S1 in Pomeranz Krummel et al., 2009Pomeranz Krummel D.A. Oubridge C. Leung A.K.W. Li J. Nagai K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution.Nature. 2009; 458: 475-480Crossref PubMed Scopus (243) Google Scholar for crystal statistics). The EMTS-soaked U1-C Q39C crystal also exhibited lower mosaic spread than the other crystals. Because the crystal was not isomorphous with those used to calculate the original maps, we used multi-NCS, multi-crystal averaging to take advantage of both the 4-fold redundancy of U1 particles in the ASU and the superior diffraction of the U1-C Q39C-EMTS.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., 1991Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models.Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar). Masks were created for each of these substructures using the program NCSMASK (CCP4, 1994Collaborative Computational Project, Number 4The CCP4 suite: programs for protein crystallography.Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19698) Google Scholar). 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., 2001Cowtan K.D. Zhang K.Y.J. Main P. DM/DMMULTI software for phase improvement by density modification.in: Rossmann M.G. Arnold E. International Tables for Crystallography, Volume F. Crystallography of Biological Macromolecules. Kluwer Academic Publishers, Dordrecht, Netherlands2001: 705-710Google Scholar). 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.Figure 3Superposition of NCS-Related ParticlesShow full captionThe four particles in the ASU were superimposed in O (Jones et al., 1991Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models.Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Accuracy of Sm Protein Selenium Positions at 5.5 Å ResolutionWe 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., 1999Kambach C. Walke S. Young R. Avis J.M. de la Fortelle E. Raker V.A. Lührmann R. Li J. Nagai K. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs.Cell. 1999; 96: 375-387Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), 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" @default.
• W2021506799 created "2016-06-24" @default.
• W2021506799 creator A5038030308 @default.
• W2021506799 creator A5050151179 @default.
• W2021506799 creator A5056271884 @default.
• W2021506799 creator A5062729861 @default.
• W2021506799 creator A5068936914 @default.
• W2021506799 date "2009-07-01" @default.
• W2021506799 modified "2023-09-27" @default.
• W2021506799 title "Interpreting a Low Resolution Map of Human U1 snRNP Using Anomalous Scatterers" @default.
• W2021506799 cites W1591680814 @default.
• W2021506799 cites W1868347993 @default.
• W2021506799 cites W1870805240 @default.
• W2021506799 cites W1873825682 @default.
• W2021506799 cites W1976977329 @default.
• W2021506799 cites W1978607217 @default.
• W2021506799 cites W1979907792 @default.
• W2021506799 cites W1993926172 @default.
• W2021506799 cites W2001641653 @default.
• W2021506799 cites W2005744558 @default.
• W2021506799 cites W2013083986 @default.
• W2021506799 cites W2013235095 @default.
• W2021506799 cites W2016391746 @default.
• W2021506799 cites W2034890717 @default.
• W2021506799 cites W2040881232 @default.
• W2021506799 cites W2060178110 @default.
• W2021506799 cites W2060419010 @default.
• W2021506799 cites W2071804506 @default.
• W2021506799 cites W2075120021 @default.
• W2021506799 cites W2078819000 @default.
• W2021506799 cites W2079711996 @default.
• W2021506799 cites W2084619201 @default.
• W2021506799 cites W2101446086 @default.
• W2021506799 cites W2110893691 @default.
• W2021506799 cites W2111274657 @default.
• W2021506799 cites W2119966574 @default.
• W2021506799 cites W2138148973 @default.
• W2021506799 cites W2143641256 @default.
• W2021506799 cites W2144414956 @default.
• W2021506799 cites W2161116298 @default.
• W2021506799 cites W4210675630 @default.
• W2021506799 doi "https://doi.org/10.1016/j.str.2009.05.009" @default.
• W2021506799 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2712673" @default.
• W2021506799 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19604473" @default.
• W2021506799 hasPublicationYear "2009" @default.
• W2021506799 type Work @default.
• W2021506799 sameAs 2021506799 @default.
• W2021506799 citedByCount "13" @default.
• W2021506799 countsByYear W20215067992012 @default.
• W2021506799 countsByYear W20215067992013 @default.
• W2021506799 countsByYear W20215067992014 @default.
• W2021506799 countsByYear W20215067992015 @default.
• W2021506799 countsByYear W20215067992016 @default.
• W2021506799 countsByYear W20215067992017 @default.
• W2021506799 countsByYear W20215067992021 @default.
• W2021506799 crossrefType "journal-article" @default.
• W2021506799 hasAuthorship W2021506799A5038030308 @default.
• W2021506799 hasAuthorship W2021506799A5050151179 @default.
• W2021506799 hasAuthorship W2021506799A5056271884 @default.
• W2021506799 hasAuthorship W2021506799A5062729861 @default.
• W2021506799 hasAuthorship W2021506799A5068936914 @default.
• W2021506799 hasBestOaLocation W20215067991 @default.
• W2021506799 hasConcept C104317684 @default.
• W2021506799 hasConcept C138268822 @default.
• W2021506799 hasConcept C154945302 @default.
• W2021506799 hasConcept C41008148 @default.
• W2021506799 hasConcept C54355233 @default.
• W2021506799 hasConcept C54458228 @default.
• W2021506799 hasConcept C67705224 @default.
• W2021506799 hasConcept C68483431 @default.
• W2021506799 hasConcept C70721500 @default.
• W2021506799 hasConcept C86803240 @default.
• W2021506799 hasConceptScore W2021506799C104317684 @default.
• W2021506799 hasConceptScore W2021506799C138268822 @default.
• W2021506799 hasConceptScore W2021506799C154945302 @default.
• W2021506799 hasConceptScore W2021506799C41008148 @default.
• W2021506799 hasConceptScore W2021506799C54355233 @default.
• W2021506799 hasConceptScore W2021506799C54458228 @default.
• W2021506799 hasConceptScore W2021506799C67705224 @default.
• W2021506799 hasConceptScore W2021506799C68483431 @default.
• W2021506799 hasConceptScore W2021506799C70721500 @default.
• W2021506799 hasConceptScore W2021506799C86803240 @default.
• W2021506799 hasIssue "7" @default.
• W2021506799 hasLocation W20215067991 @default.
• W2021506799 hasLocation W20215067992 @default.
• W2021506799 hasLocation W20215067993 @default.
• W2021506799 hasLocation W20215067994 @default.
• W2021506799 hasOpenAccess W2021506799 @default.
• W2021506799 hasPrimaryLocation W20215067991 @default.
• W2021506799 hasRelatedWork W2021506799 @default.
• W2021506799 hasRelatedWork W2039953372 @default.
• W2021506799 hasRelatedWork W2091495597 @default.
• W2021506799 hasRelatedWork W2127130540 @default.
• W2021506799 hasRelatedWork W2151112077 @default.
• W2021506799 hasRelatedWork W2805835967 @default.
• W2021506799 hasRelatedWork W4224526861 @default.
• W2021506799 hasRelatedWork W4319869769 @default.
• W2021506799 hasRelatedWork W57618549 @default.

• next