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BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE

Yıl 2022, Cilt: 8 Sayı: 2, 60 - 69, 30.12.2022
https://doi.org/10.22531/muglajsci.1174474

Öz

Small angle X-ray Scattering (SAXS) is a method for determining basic structural characteristics such as the size, shape, and surface of particles. SAXS data can be used to generate low resolution models of biomolecules faster than any other conventional experimental structural biology tool. SAXS data is mostly collected in synchrotron facilities to obtain the best scattering data possible however home source SAXS devices can also generate valuable data when properly optimized. Here, we examined sample data collection and optimization at home source SAXS beamline in terms of the concentration, purity, and duration of data acquisition. We validated that high concentration, monodisperse and ultra pure protein samples obtained by size exclusion chromatography are necessary for generating viable SAXS data using a home source beamline. At least one hour is required to generate a feasible model from SAXS data, although longer data collection times do not always translate to higher resolutions. We show that with small optimizations during data collection and analysis SAXS can characterize properties such as oligomerization, molecular mass, and overall shape of particles in solution under physiological conditions.

Destekleyen Kurum

TÜBİTAK

Proje Numarası

120Z594, 221S535, 122Z747

Teşekkür

We are greatful to Prof. Ziarek at Indiana University-Bloomington for constructive feedback and we apprecıate Ziarek Juniors for their understanding. CD acknowledges support from TÜBİTAK (Project No: 120Z594, 221S535, 122Z747). The authors acknowledge the use of the services and facilities of n2STAR-Koç University Nanofabrication and Nanocharacterization Center for Scientific and Technological Advanced Research.

Kaynakça

  • Kane, P.P., Kissel, L., Pratt, R.H. and Roy, S.C., “Elastic scattering of γ-rays and X-rays by atoms”, Physics Reports, Vol. No. 140(2), 75–159, 1986.
  • Kane, P.P., “Inelastic scattering of X-rays and gamma rays by inner shell electrons”, Physics Reports, Vol. No. 218(2) 67–139, 1992.
  • Chavas, L.M.G., “Small Angle X-Ray and Neutron Scattering from Solutions of Biological Macromolecules”, Acta Crystallographica Section D: Biological Crystallography, Vol. No. 70(4), 1175-1176, 2014.
  • Makowski, L., “Characterization of proteins with wide-angle X-ray solution scattering (WAXS)”, Journal of Structural and Functional Genomics, Vol. No. 11(1), 9–19, 2010.
  • Das, R. and Doniach, S., (Pecora, R. and Borsali, R.), Structural Studies of Proteins and Nucleic Acids in Solution Using Small Angle X-Ray Scattering (SAXS), Soft Matter Characterization, 1083-1108, 2008.
  • Pauw, B.R., “Everything Saxs: Small-angle scattering pattern collection and correction”, Journal of Physics: Condensed Matter, Vol. No. 25(38), 383201, 2013.
  • Skou, S., Gillilan, R.E. and Ando, N., “Synchrotron-based small-angle X-ray scattering of proteins in solution”, Nature Protocols, Vol. No. 9(7), 1727-1739, 2014.
  • Grant, T.D., Luft, J.R., Wolfley, J.R., Tsuruta, H., Martel, A., Montelione, G.T. and Snell, E.H., “Small angle X-ray scattering as a complementary tool for high-throughput structural studies”, Biopolymers, Vol. No. 95(8), 517-530, 2011.
  • Matthews, B. W., “X-ray crystallographic studies of proteins”, Annual Review of Physical Chemistry, Vol. No. 27(1), 493–493, 1976.
  • Valentini, E., Kikhney, A.G., Previtali, G., Jeffries, C.M. and Svergun, D.I., “SASBDB, a repository for biological small-angle scattering data”, Nucleic Acids Research, Vol. No. 43(D1), 2014.
  • Malfois, M. and Svergun, D.I., “SASCIF: An extension of core crystallographic information file for SAS”, Journal of Applied Crystallography, Vol. No. 33(3), 812–816, 2000.
  • Kachala, M., Westbrook, J. and Svergun, D., “Extension of the sascif format and its applications for data processing and deposition”, Journal of Applied Crystallography, Vol. No. 49(1), 302-310, 2016.
  • Korasick, D.A. and Tanner, J.J., “Determination of protein oligomeric structure from small-angle X-ray scattering”, Protein Science, Vol. No. 27(4), 814–824, 2018.
  • Schindler, C.E., de Vries, S.J., Sasse, A. and Zacharias, M., “SAXS data alone can generate high-quality models of protein-protein complexes”, Structure, Vol. No. 24(8), 1387–1397, 2016.
  • Gomes, G.N.W., Krzeminski, M., Namini, A., Martin, E.W., Mittag, T., Head-Gordon, T., Forman-Kay, J.D. and Gradinaru, C.C., “Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET”, Journal of the American Chemical Society, Vol. No. 142(37), 15697-15710, 2020
  • Belviso, B.D., Mangiatordi, G.F., Alberga, D., Mangini, V., Carrozzini, B. and Caliandro, R., “Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab”, Frontiers in Molecular Biosciences, Vol. No. 9, 823174, 2022.
  • Hura, G.L., Budworth, H., Dyer, K.N., Rambo, R.P., Hammel, M., McMurray, C.T. and Tainer, J.A., “Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)”, Nature Methods, Vol. No. 6(8), 606-612, 2009.
  • Dagenais, P., Desjardins, G. and Legault, P., “An integrative NMR-SAXS approach for structural determination of large rnas defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme”, Nucleic Acids Research, Vol. No. 49(20), 11959-11973, 2021.
  • Förster, S., Apostol, L. and Bras, W., “Scatter: software for the analysis of nano- and mesoscale small-angle scattering”, Journal of Applied Crystallography, Vol. No. 43(3), 639-646, 2010.
  • Pospelov, G., Van Herck, W., Burle, J., Carmona Loaiza, J.M., Durniak, C., Fisher, J.M., Ganeva, M., Yurov, D. and Wuttke, J., “Bornagain: Software for simulating and fitting grazing-incidence small-angle scattering”, Journal of Applied Crystallography, Vol. No. 53(1), 262–276, 2020.
  • Tan, L., Elkins, J.G., Davison, B.H., Kelley, E.G. and Nickels, J., “Implementation of a self-consistent slab model of bilayer structure in the sasview suite”, Journal of Applied Crystallography, Vol. No. 54(1), 363–370, 2021.
  • Ilavsky, J. and Jemian, P.R., “Irena: tool suite for modeling and analysis of small-angle scattering”, Journal of Applied Crystallography, Vol. No. 42(2), 347-353, 2009.
  • Manalastas-Cantos, K., Konarev, P.V., Hajizadeh, N.R., Kikhney, A.G., Petoukhov, M.V., Molodenskiy, D.S., Panjkovich, A., Mertens, H.D., Gruzinov, A., Borges, C. and Jeffries, C.M., “Atsas 3.0: Expanded functionality and new tools for small-angle scattering data analysis”, Journal of Applied Crystallography, Vol. No. 54(1), 343–355, 2021.
  • Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H. and Svergun, D.I., “Primus: A windows PC-based system for small-angle scattering data analysis”, Journal of Applied Crystallography, Vol. No. 36(5), 1277–1282, 2003.
  • Svergun, D. I., “Determination of the regularization parameter in indirect-transform methods using perceptual criteria”, Journal of Applied Crystallography, Vol. No. 25(4), 495–503, 1992.
  • Svergun, D.I., “Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing”, Biophysical Journal, Vol. No. 76(6), 2879–2886, 1999.
  • Franke, D. and Svergun, D. I., “DAMMIF, a program for rapidab-initioshape determination in small-angle scattering”, Journal of Applied Crystallography, Vol. No. 42(2), 342-346, 2009.
  • Svergun, D.I.B.C., Barberato, C. and Koch, M.H., “Crysol– a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates”, Journal of Applied Crystallography, Vol. No. 28(6), 768–773, 1995.
  • Mertens, H.D. and Svergun, D.I., “Combining NMR and small angle X-ray scattering for the study of biomolecular structure and Dynamics”, Archives of Biochemistry and Biophysics, Vol. No. 628, 33–41, 2017.
  • Tokgöz, Z., Siepmann, T.J., Streich, F., Kumar, B., Klein, J.M. and Haas, A.L., “E1-E2 interactions in Ubiquitin and NEDD8 ligation pathways”, Journal of Biological Chemistry, Vol. No. 287(1), 311–321, 2012.
  • Ronchi, V.P., Klein, J.M. and Haas, A.L., “E6AP/UBE3A ubiquitin ligase harbors two e2∼ubiquitin binding sites”, Journal of Biological Chemistry, Vol. No. 288(15), 10349–10360, 2013.
  • Franke, D., Petoukhov, M.V., Konarev, P.V., Panjkovich, A., Tuukkanen, A., Mertens, H.D.T., Kikhney, A.G., Hajizadeh, N.R., Franklin, J.M., Jeffries, C.M. and Svergun, D., “Atsas 2.8: A comprehensive data analysis suite for small-angle scattering from Macromolecular Solutions”, Journal of Applied Crystallography, Vol. No. 50(4), 1212–1225, 2017.
  • Živič, Z., Strmšek, Ž., Novinec, M., Lah, J. and Hadži, S., “Structural polymorphism of coiled‐coils from the stalk domain of sars‐cov‐2 Spike protein”, The FASEB Journal, Vol. No. 36(3), e22199, 2022.
  • Mylonas, E. and Svergun, D.I., “Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering”, Journal of Applied Crystallography, Vol. No. 40(S1), 245-249, 2007.
  • Burger, V.M., Arenas, D.J. and Stultz, C.M., “A Structure-free Method for Quantifying Conformational Flexibility in proteins”, Scientific Reports, Vol. No. 6(1), 1-9, 2016.
  • Putnam, C.D., Hammel, M., Hura, G.L. and Tainer, J.A., “X-ray solution scattering (SAXS) combined with crystallography and computation: Defining accurate macromolecular structures, conformations and assemblies in solution”, Quarterly Reviews of Biophysics, Vol. No. 40(3), 191–285, 2007.
  • Graewert, M.A., Da Vela, S., Gräwert, T.W., Molodenskiy, D.S., Blanchet, C.E., Svergun, D.I. and Jeffries, C.M., ”Adding SEC and Light Scattering (LS) Devices to Obtain High-Quality SAXS Data”, Crystals, 10(11), 975, 2020.
  • He, H., Liu, C. and Liu, H., “Model Reconstruction from Small-Angle X-Ray Scattering Data Using Deep Learning Methods”, Iscience, Vol. No. 23(3), 100906, 2020
  • Sayers, Z., Avşar, B., Cholak, E., & Karmous, I. “Application of advanced X-ray methods in life sciences“. Biochimica et Biophysica Acta, Vol. No. 1861(1), 3671-3685, 2017
Yıl 2022, Cilt: 8 Sayı: 2, 60 - 69, 30.12.2022
https://doi.org/10.22531/muglajsci.1174474

Öz

Proje Numarası

120Z594, 221S535, 122Z747

Kaynakça

  • Kane, P.P., Kissel, L., Pratt, R.H. and Roy, S.C., “Elastic scattering of γ-rays and X-rays by atoms”, Physics Reports, Vol. No. 140(2), 75–159, 1986.
  • Kane, P.P., “Inelastic scattering of X-rays and gamma rays by inner shell electrons”, Physics Reports, Vol. No. 218(2) 67–139, 1992.
  • Chavas, L.M.G., “Small Angle X-Ray and Neutron Scattering from Solutions of Biological Macromolecules”, Acta Crystallographica Section D: Biological Crystallography, Vol. No. 70(4), 1175-1176, 2014.
  • Makowski, L., “Characterization of proteins with wide-angle X-ray solution scattering (WAXS)”, Journal of Structural and Functional Genomics, Vol. No. 11(1), 9–19, 2010.
  • Das, R. and Doniach, S., (Pecora, R. and Borsali, R.), Structural Studies of Proteins and Nucleic Acids in Solution Using Small Angle X-Ray Scattering (SAXS), Soft Matter Characterization, 1083-1108, 2008.
  • Pauw, B.R., “Everything Saxs: Small-angle scattering pattern collection and correction”, Journal of Physics: Condensed Matter, Vol. No. 25(38), 383201, 2013.
  • Skou, S., Gillilan, R.E. and Ando, N., “Synchrotron-based small-angle X-ray scattering of proteins in solution”, Nature Protocols, Vol. No. 9(7), 1727-1739, 2014.
  • Grant, T.D., Luft, J.R., Wolfley, J.R., Tsuruta, H., Martel, A., Montelione, G.T. and Snell, E.H., “Small angle X-ray scattering as a complementary tool for high-throughput structural studies”, Biopolymers, Vol. No. 95(8), 517-530, 2011.
  • Matthews, B. W., “X-ray crystallographic studies of proteins”, Annual Review of Physical Chemistry, Vol. No. 27(1), 493–493, 1976.
  • Valentini, E., Kikhney, A.G., Previtali, G., Jeffries, C.M. and Svergun, D.I., “SASBDB, a repository for biological small-angle scattering data”, Nucleic Acids Research, Vol. No. 43(D1), 2014.
  • Malfois, M. and Svergun, D.I., “SASCIF: An extension of core crystallographic information file for SAS”, Journal of Applied Crystallography, Vol. No. 33(3), 812–816, 2000.
  • Kachala, M., Westbrook, J. and Svergun, D., “Extension of the sascif format and its applications for data processing and deposition”, Journal of Applied Crystallography, Vol. No. 49(1), 302-310, 2016.
  • Korasick, D.A. and Tanner, J.J., “Determination of protein oligomeric structure from small-angle X-ray scattering”, Protein Science, Vol. No. 27(4), 814–824, 2018.
  • Schindler, C.E., de Vries, S.J., Sasse, A. and Zacharias, M., “SAXS data alone can generate high-quality models of protein-protein complexes”, Structure, Vol. No. 24(8), 1387–1397, 2016.
  • Gomes, G.N.W., Krzeminski, M., Namini, A., Martin, E.W., Mittag, T., Head-Gordon, T., Forman-Kay, J.D. and Gradinaru, C.C., “Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET”, Journal of the American Chemical Society, Vol. No. 142(37), 15697-15710, 2020
  • Belviso, B.D., Mangiatordi, G.F., Alberga, D., Mangini, V., Carrozzini, B. and Caliandro, R., “Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab”, Frontiers in Molecular Biosciences, Vol. No. 9, 823174, 2022.
  • Hura, G.L., Budworth, H., Dyer, K.N., Rambo, R.P., Hammel, M., McMurray, C.T. and Tainer, J.A., “Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)”, Nature Methods, Vol. No. 6(8), 606-612, 2009.
  • Dagenais, P., Desjardins, G. and Legault, P., “An integrative NMR-SAXS approach for structural determination of large rnas defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme”, Nucleic Acids Research, Vol. No. 49(20), 11959-11973, 2021.
  • Förster, S., Apostol, L. and Bras, W., “Scatter: software for the analysis of nano- and mesoscale small-angle scattering”, Journal of Applied Crystallography, Vol. No. 43(3), 639-646, 2010.
  • Pospelov, G., Van Herck, W., Burle, J., Carmona Loaiza, J.M., Durniak, C., Fisher, J.M., Ganeva, M., Yurov, D. and Wuttke, J., “Bornagain: Software for simulating and fitting grazing-incidence small-angle scattering”, Journal of Applied Crystallography, Vol. No. 53(1), 262–276, 2020.
  • Tan, L., Elkins, J.G., Davison, B.H., Kelley, E.G. and Nickels, J., “Implementation of a self-consistent slab model of bilayer structure in the sasview suite”, Journal of Applied Crystallography, Vol. No. 54(1), 363–370, 2021.
  • Ilavsky, J. and Jemian, P.R., “Irena: tool suite for modeling and analysis of small-angle scattering”, Journal of Applied Crystallography, Vol. No. 42(2), 347-353, 2009.
  • Manalastas-Cantos, K., Konarev, P.V., Hajizadeh, N.R., Kikhney, A.G., Petoukhov, M.V., Molodenskiy, D.S., Panjkovich, A., Mertens, H.D., Gruzinov, A., Borges, C. and Jeffries, C.M., “Atsas 3.0: Expanded functionality and new tools for small-angle scattering data analysis”, Journal of Applied Crystallography, Vol. No. 54(1), 343–355, 2021.
  • Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H. and Svergun, D.I., “Primus: A windows PC-based system for small-angle scattering data analysis”, Journal of Applied Crystallography, Vol. No. 36(5), 1277–1282, 2003.
  • Svergun, D. I., “Determination of the regularization parameter in indirect-transform methods using perceptual criteria”, Journal of Applied Crystallography, Vol. No. 25(4), 495–503, 1992.
  • Svergun, D.I., “Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing”, Biophysical Journal, Vol. No. 76(6), 2879–2886, 1999.
  • Franke, D. and Svergun, D. I., “DAMMIF, a program for rapidab-initioshape determination in small-angle scattering”, Journal of Applied Crystallography, Vol. No. 42(2), 342-346, 2009.
  • Svergun, D.I.B.C., Barberato, C. and Koch, M.H., “Crysol– a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates”, Journal of Applied Crystallography, Vol. No. 28(6), 768–773, 1995.
  • Mertens, H.D. and Svergun, D.I., “Combining NMR and small angle X-ray scattering for the study of biomolecular structure and Dynamics”, Archives of Biochemistry and Biophysics, Vol. No. 628, 33–41, 2017.
  • Tokgöz, Z., Siepmann, T.J., Streich, F., Kumar, B., Klein, J.M. and Haas, A.L., “E1-E2 interactions in Ubiquitin and NEDD8 ligation pathways”, Journal of Biological Chemistry, Vol. No. 287(1), 311–321, 2012.
  • Ronchi, V.P., Klein, J.M. and Haas, A.L., “E6AP/UBE3A ubiquitin ligase harbors two e2∼ubiquitin binding sites”, Journal of Biological Chemistry, Vol. No. 288(15), 10349–10360, 2013.
  • Franke, D., Petoukhov, M.V., Konarev, P.V., Panjkovich, A., Tuukkanen, A., Mertens, H.D.T., Kikhney, A.G., Hajizadeh, N.R., Franklin, J.M., Jeffries, C.M. and Svergun, D., “Atsas 2.8: A comprehensive data analysis suite for small-angle scattering from Macromolecular Solutions”, Journal of Applied Crystallography, Vol. No. 50(4), 1212–1225, 2017.
  • Živič, Z., Strmšek, Ž., Novinec, M., Lah, J. and Hadži, S., “Structural polymorphism of coiled‐coils from the stalk domain of sars‐cov‐2 Spike protein”, The FASEB Journal, Vol. No. 36(3), e22199, 2022.
  • Mylonas, E. and Svergun, D.I., “Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering”, Journal of Applied Crystallography, Vol. No. 40(S1), 245-249, 2007.
  • Burger, V.M., Arenas, D.J. and Stultz, C.M., “A Structure-free Method for Quantifying Conformational Flexibility in proteins”, Scientific Reports, Vol. No. 6(1), 1-9, 2016.
  • Putnam, C.D., Hammel, M., Hura, G.L. and Tainer, J.A., “X-ray solution scattering (SAXS) combined with crystallography and computation: Defining accurate macromolecular structures, conformations and assemblies in solution”, Quarterly Reviews of Biophysics, Vol. No. 40(3), 191–285, 2007.
  • Graewert, M.A., Da Vela, S., Gräwert, T.W., Molodenskiy, D.S., Blanchet, C.E., Svergun, D.I. and Jeffries, C.M., ”Adding SEC and Light Scattering (LS) Devices to Obtain High-Quality SAXS Data”, Crystals, 10(11), 975, 2020.
  • He, H., Liu, C. and Liu, H., “Model Reconstruction from Small-Angle X-Ray Scattering Data Using Deep Learning Methods”, Iscience, Vol. No. 23(3), 100906, 2020
  • Sayers, Z., Avşar, B., Cholak, E., & Karmous, I. “Application of advanced X-ray methods in life sciences“. Biochimica et Biophysica Acta, Vol. No. 1861(1), 3671-3685, 2017
Toplam 39 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Bölüm Makaleler
Yazarlar

Oktay Göcenler 0000-0003-4310-0928

Cansu Müşerref Yenici 0000-0003-0179-6269

Kerem Kahraman 0000-0002-7992-5908

Cengizhan Büyükdağ 0000-0001-9714-4892

Çağdaş Dağ 0000-0003-1595-431X

Proje Numarası 120Z594, 221S535, 122Z747
Erken Görünüm Tarihi 2 Kasım 2022
Yayımlanma Tarihi 30 Aralık 2022
Yayımlandığı Sayı Yıl 2022 Cilt: 8 Sayı: 2

Kaynak Göster

APA Göcenler, O., Yenici, C. M., Kahraman, K., Büyükdağ, C., vd. (2022). BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE. Mugla Journal of Science and Technology, 8(2), 60-69. https://doi.org/10.22531/muglajsci.1174474
AMA Göcenler O, Yenici CM, Kahraman K, Büyükdağ C, Dağ Ç. BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE. Mugla Journal of Science and Technology. Aralık 2022;8(2):60-69. doi:10.22531/muglajsci.1174474
Chicago Göcenler, Oktay, Cansu Müşerref Yenici, Kerem Kahraman, Cengizhan Büyükdağ, ve Çağdaş Dağ. “BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE”. Mugla Journal of Science and Technology 8, sy. 2 (Aralık 2022): 60-69. https://doi.org/10.22531/muglajsci.1174474.
EndNote Göcenler O, Yenici CM, Kahraman K, Büyükdağ C, Dağ Ç (01 Aralık 2022) BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE. Mugla Journal of Science and Technology 8 2 60–69.
IEEE O. Göcenler, C. M. Yenici, K. Kahraman, C. Büyükdağ, ve Ç. Dağ, “BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE”, Mugla Journal of Science and Technology, c. 8, sy. 2, ss. 60–69, 2022, doi: 10.22531/muglajsci.1174474.
ISNAD Göcenler, Oktay vd. “BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE”. Mugla Journal of Science and Technology 8/2 (Aralık 2022), 60-69. https://doi.org/10.22531/muglajsci.1174474.
JAMA Göcenler O, Yenici CM, Kahraman K, Büyükdağ C, Dağ Ç. BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE. Mugla Journal of Science and Technology. 2022;8:60–69.
MLA Göcenler, Oktay vd. “BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE”. Mugla Journal of Science and Technology, c. 8, sy. 2, 2022, ss. 60-69, doi:10.22531/muglajsci.1174474.
Vancouver Göcenler O, Yenici CM, Kahraman K, Büyükdağ C, Dağ Ç. BIOMOLECULAR SOLUTION X-RAY SCATTERING AT n2STAR BEAMLINE. Mugla Journal of Science and Technology. 2022;8(2):60-9.

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