Title: Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex
Abstract: The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup13355–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup13355–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. The Saccharomyces cerevisiae nuclear pore complex (NPC) 1The abbreviations used are:NPCnuclear pore complexSAXSsmall angle X-ray scatteringEMelectron microscopynupnucleoporinScSaccharomyces cerevisiaeVpVanderwaltozyma polysporaHsHomo sapiensALPSArfGAP1 lipid packing sensorGFPgreen fluorescent proteinNEnuclear envelopeDSSdisuccinimidyl suberateEDC1-ethyl-3-(3-dimethylaminopropyl) carbodiimideSeMET-SADSelenomethionine Single-wavelength Anomalous Dispersion. 1The abbreviations used are:NPCnuclear pore complexSAXSsmall angle X-ray scatteringEMelectron microscopynupnucleoporinScSaccharomyces cerevisiaeVpVanderwaltozyma polysporaHsHomo sapiensALPSArfGAP1 lipid packing sensorGFPgreen fluorescent proteinNEnuclear envelopeDSSdisuccinimidyl suberateEDC1-ethyl-3-(3-dimethylaminopropyl) carbodiimideSeMET-SADSelenomethionine Single-wavelength Anomalous Dispersion. is a large macromolecular assembly of ∼50 MDa made of at least 456 protein copies of ∼30 distinct proteins called nucleoporins (nups). The NPC is the sole passageway for the exchange of macromolecules across the nuclear envelope (NE) (1Aitchison J.D. Rout M.P. The yeast nuclear pore complex and transport through it.Genetics. 2012; 190: 855-883Crossref PubMed Scopus (105) Google Scholar). Apart from its main function as the sole mediator of nucleocytoplasmic trafficking, the NPC plays additional roles in numerous essential cellular processes, such as gene expression and chromatin regulation (2Wozniak R. Burke B. Doye V. Nuclear transport and the mitotic apparatus: an evolving relationship.Cell Mol. Life Sci. 2010; 67: 2215-2230Crossref PubMed Scopus (79) Google Scholar), and defects in its components have been implicated in numerous major human diseases (3Grossman E. Medalia O. Zwerger M. Functional architecture of the nuclear pore complex.Annu. Rev. Biophys. 2012; 41: 557-584Crossref PubMed Scopus (192) Google Scholar). The first description of the macromolecular architecture of the NPC (4Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (818) Google Scholar) was determined via an integrative approach based on a wide variety of experimental data (5Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Rout M.P. Sali A. Determining the architectures of macromolecular assemblies.Nature. 2007; 450: 683-694Crossref PubMed Scopus (438) Google Scholar). The permeability barrier is formed by FG (phenylalanine-glycine repeat–containing) nups, which fill the central channel of the NPC and are anchored to the core scaffold (6Peters R. Translocation through the nuclear pore: Kaps pave the way.Bioessays. 2009; 31: 466-477Crossref PubMed Scopus (95) Google Scholar). The NPC architectural core is formed by an 8-fold arrangement of symmetric units called spokes that connect to each other, forming coaxial rings: two outer rings (the nuclear and cytoplasmic rings), a membrane ring, and two inner rings (7Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. The yeast nuclear pore complex: composition, architecture, and transport mechanism.J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1149) Google Scholar). In S. cerevisiae, the membrane ring is mainly formed by the transmembrane nups Pom152, Pom34, and Ndc1; the two adjacent inner rings are formed by large nups Nup192, Nup188, Nup170, and Nup157; and the two outer rings are formed by a radial head-to-tail arrangement of eight copies of the Nup84 complex (4Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (818) Google Scholar, 8Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R. Stokes D.L. Chait B.T. Sali A. Rout M.P. Structure-function mapping of a heptameric module in the nuclear pore complex.J. Cell Biol. 2012; 196: 419-434Crossref PubMed Scopus (90) Google Scholar, 9Szymborska A. de Marco A. Daigle N. Cordes V.C. Briggs J.A. Ellenberg J. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.Science. 2013; 341: 655-658Crossref PubMed Scopus (296) Google Scholar). The Nup84 complex is a conserved assembly formed by nine proteins in vertebrates (Nup107–160 complex) and by seven nups in yeast (Nup133, Nup120, Nup145c, Nup85, Nup84, Seh1, and Sec13). The yeast Nup84 complex arranges into a characteristic Y-shaped assembly (10Lutzmann M. Kunze R. Buerer A. Aebi U. Hurt E. Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins.EMBO J. 2002; 21: 387-397Crossref PubMed Scopus (178) Google Scholar, 11Kampmann M. Atkinson C.E. Mattheyses A.L. Simon S.M. Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy.Nat. Struct. Mol. Biol. 2011; 18: 643-649Crossref PubMed Scopus (59) Google Scholar). The stalk of the Y is formed by a tail-to-tail connection between Nup133 and Nup84 and a head-to-center connection between Nup84 and the dimer Nup145c-Sec13 (8Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R. Stokes D.L. Chait B.T. Sali A. Rout M.P. Structure-function mapping of a heptameric module in the nuclear pore complex.J. Cell Biol. 2012; 196: 419-434Crossref PubMed Scopus (90) Google Scholar, 12Seo H.S. Ma Y. Debler E.W. Wacker D. Kutik S. Blobel G. Hoelz A. Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14281-14286Crossref PubMed Scopus (62) Google Scholar, 13Bui K.H. von Appen A. DiGuilio A.L. Ori A. Sparks L. Mackmull M.T. Bock T. Hagen W. Andres-Pons A. Glavy J.S. Beck M. Integrated structural analysis of the human nuclear pore complex scaffold.Cell. 2013; 155: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). nuclear pore complex small angle X-ray scattering electron microscopy nucleoporin Saccharomyces cerevisiae Vanderwaltozyma polyspora Homo sapiens ArfGAP1 lipid packing sensor green fluorescent protein nuclear envelope disuccinimidyl suberate 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide Selenomethionine Single-wavelength Anomalous Dispersion. nuclear pore complex small angle X-ray scattering electron microscopy nucleoporin Saccharomyces cerevisiae Vanderwaltozyma polyspora Homo sapiens ArfGAP1 lipid packing sensor green fluorescent protein nuclear envelope disuccinimidyl suberate 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide Selenomethionine Single-wavelength Anomalous Dispersion. Nup133, a 133-kDa subunit of the Nup84 complex, consists of an N-terminal β-propeller and a C-terminal α-solenoid-like folds (14Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Simple fold composition and modular architecture of the nuclear pore complex.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2172-2177Crossref PubMed Scopus (225) Google Scholar). Nup133 is located at the end of the stalk of the Nup84 complex through a connection with Nup84 (8Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R. Stokes D.L. Chait B.T. Sali A. Rout M.P. Structure-function mapping of a heptameric module in the nuclear pore complex.J. Cell Biol. 2012; 196: 419-434Crossref PubMed Scopus (90) Google Scholar). Nup133 has also been suggested to connect through the first 15 residues of its N-terminal domain to the Nup120 copy of an adjacent Nup84 complex heptamer (12Seo H.S. Ma Y. Debler E.W. Wacker D. Kutik S. Blobel G. Hoelz A. Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14281-14286Crossref PubMed Scopus (62) Google Scholar). Nup133 is a highly conserved nup that plays key roles in interphase and post-mitotic NPC biogenesis (15Doucet C.M. Talamas J.A. Hetzer M.W. Cell cycle-dependent differences in nuclear pore complex assembly in metazoa.Cell. 2010; 141: 1030-1041Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 16Walther T.C. Alves A. Pickersgill H. Loiodice I. Hetzer M. Galy V. Hulsmann B.B. Kocher T. Wilm M. Allen T. Mattaj I.W. Doye V. The conserved Nup107–160 complex is critical for nuclear pore complex assembly.Cell. 2003; 113: 195-206Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar), as well as in efficient anchoring of the dynein/dynactin complex to tether centrosomes to the NE in prophase (17Bolhy S. Bouhlel I. Dultz E. Nayak T. Zuccolo M. Gatti X. Vallee R. Ellenberg J. Doye V. A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase.J. Cell Biol. 2011; 192: 855-871Crossref PubMed Scopus (134) Google Scholar). A loop within the N-terminal β-propeller of human Nup133 was suggested to contain an ArfGAP1 lipid packing sensor (ALPS) motif (18Drin G. Casella J.F. Gautier R. Boehmer T. Schwartz T.U. Antonny B. A general amphipathic alpha-helical motif for sensing membrane curvature.Nat. Struct. Mol. Biol. 2007; 14: 138-146Crossref PubMed Scopus (439) Google Scholar), functioning as a membrane curvature sensor. This motif allows human Nup133 to interact with curved membranes both in vitro and in vivo (15Doucet C.M. Talamas J.A. Hetzer M.W. Cell cycle-dependent differences in nuclear pore complex assembly in metazoa.Cell. 2010; 141: 1030-1041Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 18Drin G. Casella J.F. Gautier R. Boehmer T. Schwartz T.U. Antonny B. A general amphipathic alpha-helical motif for sensing membrane curvature.Nat. Struct. Mol. Biol. 2007; 14: 138-146Crossref PubMed Scopus (439) Google Scholar) and has been shown to be required for proper NPC biogenesis during interphase (15Doucet C.M. Talamas J.A. Hetzer M.W. Cell cycle-dependent differences in nuclear pore complex assembly in metazoa.Cell. 2010; 141: 1030-1041Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). However, previous studies have not been able to detect any membrane interaction motifs in yeast Nup133, leading to the suggestion that the ALPS motif in Nup133 is unique to organisms with open mitosis (18Drin G. Casella J.F. Gautier R. Boehmer T. Schwartz T.U. Antonny B. A general amphipathic alpha-helical motif for sensing membrane curvature.Nat. Struct. Mol. Biol. 2007; 14: 138-146Crossref PubMed Scopus (439) Google Scholar, 19Berke I.C. Boehmer T. Blobel G. Schwartz T.U. Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.J. Cell Biol. 2004; 167: 591-597Crossref PubMed Scopus (94) Google Scholar), in turn implying that the ALPS motif is not even a part of the mechanism for membrane association of the NPCs in all eukaryotes. Interestingly, mutations in S. cerevisiae (Sc) Nup133 cause a characteristic phenotype that leads to clustering of the NPCs into discrete regions of the NE (20Doye V. Wepf R. Hurt E.C. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.EMBO J. 1994; 13: 6062-6075Crossref PubMed Scopus (207) Google Scholar). Structure–function mapping of this NPC clustering phenotype suggests that ScNup133—as well as its ancient paralog ScNup120—is functionally involved in the stabilization of the NE membrane curvature (8Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R. Stokes D.L. Chait B.T. Sali A. Rout M.P. Structure-function mapping of a heptameric module in the nuclear pore complex.J. Cell Biol. 2012; 196: 419-434Crossref PubMed Scopus (90) Google Scholar), although the exact mechanism that drives the interaction of these proteins with the NE is unknown. Multi-domain, full-length nucleoporins are generally not amenable to X-ray crystallographic structure determination, presumably because of their apparent flexibility. Indeed, the structures of the N- and C-terminal fragments of Nup133 in particular were determined only separately (19Berke I.C. Boehmer T. Blobel G. Schwartz T.U. Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.J. Cell Biol. 2004; 167: 591-597Crossref PubMed Scopus (94) Google Scholar, 21Sampathkumar P. Gheyi T. Miller S.A. Bain K.T. Dickey M. Bonanno J.B. Kim S.J. Phillips J. Pieper U. Fernandez-Martinez J. Franke J.D. Martel A. Tsuruta H. Atwell S. Thompson D.A. Emtage J.S. Wasserman S.R. Rout M.P. Sali A. Sauder J.M. Burley S.K. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex.Proteins. 2011; 79: 1672-1677Crossref PubMed Scopus (14) Google Scholar, 22Whittle J.R. Schwartz T.U. Architectural nucleoporins Nup157/170 and Nup133 are structurally related and descend from a second ancestral element.J. Biol. Chem. 2009; 284: 28442-28452Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 23Boehmer T. Jeudy S. Berke I.C. Schwartz T.U. Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex.Mol. Cell. 2008; 30: 721-731Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); the full-length atomic structure has not yet been characterized. Consequently, the relative orientation of the N- and C-terminal domains was depicted only schematically (22Whittle J.R. Schwartz T.U. Architectural nucleoporins Nup157/170 and Nup133 are structurally related and descend from a second ancestral element.J. Biol. Chem. 2009; 284: 28442-28452Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We therefore took an integrative approach to generate the structure and dynamics of full-length ScNup133, based on multiple types of data. Here, we characterized the configuration of the individual domains, defining the shape and populations of the full-length ScNup133 conformations, based on template structures, X-ray crystallography, small angle X-ray scattering (SAXS), and electron microscopy (EM) data, and performed validation with mutational studies and a dataset from chemical cross-linking with mass spectrometric readouts. More specifically, we report the crystal structure of the Nup133 N-terminal domain (residues 55–502) from Vanderwaltozyma polyspora (Vp), as well as SAXS profiles for 18 constructs of ScNup133 and one VpNup133 construct and 23 negative-stain EM class averages of ScNup1332–1157. Using our integrative modeling approach described in this study, we then determined atomic models for multiple states of the full-length ScNup133, based on these new data as well as known structures of ScNup133944–1157 and a number of Nup133 homologs. The resulting model was subsequently validated by three sets of double point mutations at the ScNup133–ScNup84 interface and 20 disuccinimidyl suberate (DSS) and 25 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemical cross-links determined via mass spectrometry (24Shi Y. Fernandez-Martinez J. Tjioe E. Pellarin R. Kim S.J. Williams R. Schneidman-Duhovny D. Sali A. Rout M.P. Chait B.T. Structural characterization by cross-linking reveals the detailed architecture of a coatomer-related heptameric module from the nuclear pore complex.Mol Cell Proteomics. 2014; (mcp.M114.041673. First Published on August 26, 2014, doi: 10.1074/mcp.M114.041673)Abstract Full Text Full Text PDF Scopus (119) Google Scholar). As a result, the model of the full-length ScNup133 allows us to annotate a potential ALPS motif in Sc- and VpNup133, suggesting that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the NE. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. Nup133 is divided into the N-terminal β-propeller and the C-terminal α-solenoid domains in an iterative manual process relying on predicted secondary structure, gaps in multiple sequence alignments, and sequence–structure alignment by threading (14Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Simple fold composition and modular architecture of the nuclear pore complex.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2172-2177Crossref PubMed Scopus (225) Google Scholar). We cloned, expressed, and purified the resulting 18 constructs of ScNup133: 7 constructs covering the N-terminal domain, 8 constructs covering the C-terminal domain, and 3 constructs covering both domains partially or entirely (supplemental Fig. S3 and supplemental Table S1). Cloning, expression, and purification were performed using a standard protocol as described previously (21Sampathkumar P. Gheyi T. Miller S.A. Bain K.T. Dickey M. Bonanno J.B. Kim S.J. Phillips J. Pieper U. Fernandez-Martinez J. Franke J.D. Martel A. Tsuruta H. Atwell S. Thompson D.A. Emtage J.S. Wasserman S.R. Rout M.P. Sali A. Sauder J.M. Burley S.K. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex.Proteins. 2011; 79: 1672-1677Crossref PubMed Scopus (14) Google Scholar) (supplemental "Experimental Procedures" section). The N-terminal domain of V. polyspora Nup133 covering residues 55 to 502 (VpNup13355–502) was also cloned, expressed, and purified following similar procedures (21Sampathkumar P. Gheyi T. Miller S.A. Bain K.T. Dickey M. Bonanno J.B. Kim S.J. Phillips J. Pieper U. Fernandez-Martinez J. Franke J.D. Martel A. Tsuruta H. Atwell S. Thompson D.A. Emtage J.S. Wasserman S.R. Rout M.P. Sali A. Sauder J.M. Burley S.K. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex.Proteins. 2011; 79: 1672-1677Crossref PubMed Scopus (14) Google Scholar). The crystal used for structure determination via SeMET-SAD phasing was obtained by means of sitting-drop vapor diffusion (VpNup13355–502 concentration of 10.6 mg/ml) in the presence of 10% PEG3350, 100 mm ammonium sulfate, and 100 mm HEPES (pH 8.2) and flash-frozen in liquid nitrogen with 30% (v/v) glycerol. The diffraction dataset collected at the LRL-CAT 31-ID (Advanced Photon Source) beamline was processed with XDS (25Kabsch W. XDS.Acta Crystallogr. D. 2010; 66: 125-132Crossref PubMed Scopus (11228) Google Scholar) and AIMLESS (26Evans P.R. Murshudov G.N. How good are my data and what is the resolution?.Acta Crystallogr. D. 2013; 69: 1204-1214Crossref PubMed Scopus (2685) Google Scholar), and structure solution was obtained using AutoSol (27Terwilliger T.C. Adams P.D. Read R.J. McCoy A.J. Moriarty N.W. Grosse-Kunstleve R.W. Afonine P.V. Zwart P.H. Hung L.W. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard.Acta Crystallogr. 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SAXS profiles of 18 constructs of ScNup133 and one construct of VpNup13355–502(Figs. 2B and 3B, supplemental Figs. S3 and S4, and supplemental Table S1) were measured at concentrations of 0.5, 1.0, and 2.0 mg/ml and the highest possible concentrations in the protein storage buffer at 10 °C to 15 °C, using up to 24 1-s to 10-s exposures at the SSRL (Menlo Park, CA) and ALS (Berkeley, CA) beamlines (supplemental "Experimental Procedures" section). The buffer SAXS profile was obtained in the same manner and subtracted from a protein SAXS profile. The merged experimental SAXS profile of VpNup13355–502 was compared with SAXS profiles calculated using FoXS (38Schneidman-Duhovny D. Hammel M. Sali A. FoXS: a web server for rapid computation and fitting of SAXS profiles.Nucleic Acids Res. 2010; 38: W540-W544Crossref PubMed Scopus (395) Google Scholar, 39Schneidman-Duhovny D. Hammel M. Tainer J.A. Sali A. Accurate SAXS profile computation and its assessment by contrast variation experiments.Biophys. J. 2013; 105: 962-974Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), for the crystal structure of VpNup13355–502 and the "complete" models in which disordered components and four Se-Met residues were built using MODELLER 9.13 (40Sali A. Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints.J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10512) Google Scholar) (Fig. 2B).Fig. 3Structure and dynamics of ScNup133 revealed through integrative modeling approach. A, the minimal ensemble of four conformations (the multi-state model), comprising a single major extended conformation with a population weight of 0.506 (blue) and three minor compact conformations with weights of 0.242 (red), 0.202 (cyan), and 0.050 (yellow), is shown. The most populated conformation (blue) was used as a reference for rigid body least-squares superposition of the remaining three conformations. The ab initio shape (represented as a gray envelope) computed from the experimental SAXS profile was also superposed for comparison. B, comparison of the merged experimental SAXS profile (black) of ScNup1332–1157 with the calculated SAXS profiles from the ScNup1332–1157 comparative model (χ = 6.27, red) and the ensemble of four conformations (χ = 1.54, blue). The lower plot presents the residuals (calculated intensity/experimental intensity) of each calculated SAXS profile. The upper inset shows the SAXS profiles in the Guinier plot with an Rg fit of 48.3 ± 0.6 Å. The maximum particle size (Dmax) was 169.2 Å (determined experimentally). C, the 23 negative-stain EM class averages are shown along with the projections of each of the four conformations. 22 EM class averages were assigned to at least one of the four conformations with high confidence, as highlighted in colored boxes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A specimen of the full-length ScNup1332–1157 was prepared for negative-stain EM (41Ohi M. Li Y. Cheng Y. Walz T. Negative staining and image classification—powerful tools in modern electron microscopy.Biol. Proc. Online. 2004; 6: 23-34Crossref PubMed Scopus (500) Google Scholar) (supplemental "Experimental Procedures" section). The 1976 individual particles were selected interactively from images using Boxer from EMAN (42Ludtke S.J. Baldwin P.R. Chiu W. EMAN: semiautomated software for high-resolution single-particle reconstructions.J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2101) Google Scholar) and windowed into individual images with a size of 120 × 120 pixels. The particles were centered and normalized and then subjected to the ISAC (iterative stable alignment and clustering) (43Yang Z. Fang J. Chittuluru J. Asturias F.J. Penczek P.A. Iterative stable alignment and clustering of 2D transmission electron microscope images.Structure. 2012; 20: 237-247Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) technique to produce 23 stable two-dimensional class averages after 10 generations (these class averages comprised 1530 of the 1976 particles) (top rows in Fig. 3C and column 2 in supplemental Table S3). We developed an integrative modeling approach that produces atomic models for multiple states of a protein based on EM images of the protein as well as SAXS profiles and crystal structures of the sequence segments and their hom