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Universally Accessible Structural Data on Macromolecular Conformation, Assembly, and Dynamics by Small Angle X-Ray Scattering for DNA Repair Insights

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DNA Damage Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2444))

Abstract

Structures provide a critical breakthrough step for biological analyses, and small angle X-ray scattering (SAXS) is a powerful structural technique to study dynamic DNA repair proteins. As toxic and mutagenic repair intermediates need to be prevented from inadvertently harming the cell, DNA repair proteins often chaperone these intermediates through dynamic conformations, coordinated assemblies, and allosteric regulation. By measuring structural conformations in solution for both proteins, DNA, RNA, and their complexes, SAXS provides insight into initial DNA damage recognition, mechanisms for validation of their substrate, and pathway regulation. Here, we describe exemplary SAXS analyses of a DNA damage response protein spanning from what can be derived directly from the data to obtaining super resolution through the use of SAXS selection of atomic models. We outline strategies and tactics for practical SAXS data collection and analysis. Making these structural experiments in reach of any basic and clinical researchers who have protein, SAXS data can readily be collected at government-funded synchrotrons, typically at no cost for academic researchers. In addition to discussing how SAXS complements and enhances cryo-electron microscopy, X-ray crystallography, NMR, and computational modeling, we furthermore discuss taking advantage of recent advances in protein structure prediction in combination with SAXS analysis.

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References

  1. Tsutakawa SE, Tsai CL, Yan C, Bralic A, Chazin WJ, Hamdan SM, Scharer OD, Ivanov I, Tainer JA (2020) Envisioning how the prototypic molecular machine TFIIH functions in transcription initiation and DNA repair. DNA Repair (Amst) 96:102972. https://doi.org/10.1016/j.dnarep.2020.102972

    Article  Google Scholar 

  2. Wilson SH, Kunkel TA (2000) Passing the baton in base excision repair. Nat Struct Biol 7(3):176–178. https://doi.org/10.1038/73260

    Article  CAS  PubMed  Google Scholar 

  3. Parikh SS, Mol CD, Hosfield DJ, Tainer JA (1999) Envisioning the molecular choreography of DNA base excision repair. Curr Opin Struct Biol 9(1):37–47. https://doi.org/10.1016/s0959-440x(99)80006-2

    Article  CAS  PubMed  Google Scholar 

  4. Mol CD, Izumi T, Mitra S, Tainer JA (2000) DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected]. Nature 403(6768):451–456. https://doi.org/10.1038/35000249

    Article  CAS  PubMed  Google Scholar 

  5. Hitomi K, Iwai S, Tainer JA (2007) The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and repair. DNA Repair 6(4):410–428. https://doi.org/10.1016/j.dnarep.2006.10.004

    Article  CAS  PubMed  Google Scholar 

  6. Krokan HE, Bjoras M (2013) Base excision repair. Cold Spring Harb Perspect Biol 5(4):a012583. https://doi.org/10.1101/cshperspect.a012583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tsutakawa SE, Sarker AH, Ng C, Arvai AS, Shin DS, Shih B, Jiang S, Thwin AC, Tsai MS, Willcox A, Her MZ, Trego KS, Raetz AG, Rosenberg D, Bacolla A, Hammel M, Griffith JD, Cooper PK, Tainer JA (2020) Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations. Proc Natl Acad Sci U S A 117(25):14127–14138. https://doi.org/10.1073/pnas.1921311117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shibata A, Moiani D, Arvai AS, Perry J, Harding SM, Genois MM, Maity R, van Rossum-Fikkert S, Kertokalio A, Romoli F, Ismail A, Ismalaj E, Petricci E, Neale MJ, Bristow RG, Masson JY, Wyman C, Jeggo PA, Tainer JA (2014) DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. Mol Cell 53(1):7–18. https://doi.org/10.1016/j.molcel.2013.11.003

    Article  CAS  PubMed  Google Scholar 

  9. Perry JJ, Yannone SM, Holden LG, Hitomi C, Asaithamby A, Han S, Cooper PK, Chen DJ, Tainer JA (2006) WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat Struct Mol Biol 13(5):414–422. https://doi.org/10.1038/nsmb1088

    Article  CAS  PubMed  Google Scholar 

  10. Tsutakawa SE, Lafrance-Vanasse J, Tainer JA (2014) The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once. DNA Repair (Amst) 19:95–107. https://doi.org/10.1016/j.dnarep.2014.03.022

    Article  CAS  Google Scholar 

  11. Holton JM, Classen S, Frankel KA, Tainer JA (2014) The R-factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures. FEBS J 281(18):4046–4060. https://doi.org/10.1111/febs.12922

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fowler NJ, Sljoka A, Williamson MP (2020) A method for validating the accuracy of NMR protein structures. Nat Commun 11(1):6321. https://doi.org/10.1038/s41467-020-20177-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hegde PM, Dutta A, Sengupta S, Mitra J, Adhikari S, Tomkinson AE, Li GM, Boldogh I, Hazra TK, Mitra S, Hegde ML (2015) THE C-terminal domain (CTD) of human DNA glycosylase NEIL1 is required for forming BERosome repair complex with DNA replication proteins at THE replicating genome dominant negative function of the CTD. J Biol Chem 290(34):20919–20933. https://doi.org/10.1074/jbc.M115.642918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hegde ML, Tsutakawa SE, Hegde PM, Holthauzen LM, Li J, Oezguen N, Hilser VJ, Tainer JA, Mitra S (2013) The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions. J Mol Biol 425(13):2359–2371. https://doi.org/10.1016/j.jmb.2013.03.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bacolla A, Sengupta S, Ye Z, Yang C, Mitra J, De-Paula RB, Hegde ML, Ahmed Z, Mort M, Cooper DN, Mitra S, Tainer JA (2021) Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin. Nucleic Acids Res 49(1):221–243. https://doi.org/10.1093/nar/gkaa1120

    Article  CAS  PubMed  Google Scholar 

  16. Williams GJ, Williams RS, Williams JS, Moncalian G, Arvai AS, Limbo O, Guenther G, SilDas S, Hammel M, Russell P, Tainer JA (2011) ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair. Nat Struct Mol Biol 18(4):423–431. https://doi.org/10.1038/nsmb.2038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Deshpande RA, Williams GJ, Limbo O, Williams RS, Kuhnlein J, Lee JH, Classen S, Guenther G, Russell P, Tainer JA, Paull TT (2014) ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J 33(5):482–500. https://doi.org/10.1002/embj.201386100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tsutakawa SE, Van Wynsberghe AW, Freudenthal BD, Weinacht CP, Gakhar L, Washington MT, Zhuang Z, Tainer JA, Ivanov I (2011) Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA. Proc Natl Acad Sci U S A 108(43):17672–17677. https://doi.org/10.1073/pnas.1110480108

    Article  PubMed  PubMed Central  Google Scholar 

  19. Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95(8):559–571. https://doi.org/10.1002/bip.21638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hammel M, Rosenberg DJ, Bierma J, Hura GL, Thapar R, Lees-Miller SP, Tainer JA (2020) Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM. Prog Biophys Mol Biol 163:74–86. https://doi.org/10.1016/j.pbiomolbio.2020.09.003

    Article  CAS  PubMed  Google Scholar 

  21. Tsutakawa SE, Shin DS, Mol CD, Izumi T, Arvai AS, Mantha AK, Szczesny B, Ivanov IN, Hosfield DJ, Maiti B, Pique ME, Frankel KA, Hitomi K, Cunningham RP, Mitra S, Tainer JA (2013) Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes. J Biol Chem 288(12):8445–8455. https://doi.org/10.1074/jbc.M112.422774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Parikh SS, Mol CD, Slupphaug G, Bharati S, Krokan HE, Tainer JA (1998) Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J 17(17):5214–5226. https://doi.org/10.1093/emboj/17.17.5214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mullins EA, Rodriguez AA, Bradley NP, Eichman BF (2019) Emerging roles of DNA glycosylases and the base excision repair pathway. Trends Biochem Sci 44(9):765–781. https://doi.org/10.1016/j.tibs.2019.04.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tsutakawa SE, Sarker AH (2021) Breaking the rules: protein sculpting in NEIL2 regulation. Structure 29(1):1–2. https://doi.org/10.1016/j.str.2020.12.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tubbs JL, Latypov V, Kanugula S, Butt A, Melikishvili M, Kraehenbuehl R, Fleck O, Marriott A, Watson AJ, Verbeek B, McGown G, Thorncroft M, Santibanez-Koref MF, Millington C, Arvai AS, Kroeger MD, Peterson LA, Williams DM, Fried MG, Margison GP, Pegg AE, Tainer JA (2009) Flipping of alkylated DNA damage bridges base and nucleotide excision repair. Nature 459(7248):808–813. https://doi.org/10.1038/nature08076

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Latypov VF, Tubbs JL, Watson AJ, Marriott AS, McGown G, Thorncroft M, Wilkinson OJ, Senthong P, Butt A, Arvai AS, Millington CL, Povey AC, Williams DM, Santibanez-Koref MF, Tainer JA, Margison GP (2012) Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines. Mol Cell 47(1):50–60. https://doi.org/10.1016/j.molcel.2012.04.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Grasby JA, Finger LD, Tsutakawa SE, Atack JM, Tainer JA (2012) Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases. Trends Biochem Sci 37(2):74–84. https://doi.org/10.1016/j.tibs.2011.10.003

    Article  CAS  PubMed  Google Scholar 

  28. Rashid F, Harris PD, Zaher MS, Sobhy MA, Joudeh LI, Yan C, Piwonski H, Tsutakawa SE, Ivanov I, Tainer JA, Habuchi S, Hamdan SM (2017) Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1. Elife 6:e21884. https://doi.org/10.7554/eLife.21884

    Article  PubMed  PubMed Central  Google Scholar 

  29. Tsutakawa SE, Classen S, Chapados BR, Arvai AS, Finger LD, Guenther G, Tomlinson CG, Thompson P, Sarker AH, Shen B, Cooper PK, Grasby JA, Tainer JA (2011) Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily. Cell 145(2):198–211. https://doi.org/10.1016/j.cell.2011.03.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Syed A, Tainer JA (2018) The MRE11-RAD50-NBS1 complex conducts the orchestration of damage signaling and outcomes to stress in DNA replication and repair. Annu Rev Biochem 87:263–294. https://doi.org/10.1146/annurev-biochem-062917-012415

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kashammer L, Saathoff JH, Lammens K, Gut F, Bartho J, Alt A, Kessler B, Hopfner KP (2019) Mechanism of DNA end sensing and processing by the Mre11-Rad50 complex. Mol Cell 76(3):382–394. e386. https://doi.org/10.1016/j.molcel.2019.07.035

    Article  CAS  PubMed  Google Scholar 

  32. Wang JL, Duboc C, Wu Q, Ochi T, Liang S, Tsutakawa SE, Lees-Miller SP, Nadal M, Tainer JA, Blundell TL, Strick TR (2018) Dissection of DNA double-strand-break repair using novel single-molecule forceps. Nat Struct Mol Biol 25(6):482–487. https://doi.org/10.1038/s41594-018-0065-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Carney SM, Moreno AT, Piatt SC, Cisneros-Aguirre M, Lopezcolorado FW, Stark JM, Loparo JJ (2020) XLF acts as a flexible connector during non-homologous end joining. Elife 9:e61920. https://doi.org/10.7554/eLife.61920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Conlin MP, Reid DA, Small GW, Chang HH, Watanabe G, Lieber MR, Ramsden DA, Rothenberg E (2017) DNA ligase IV guides end-processing choice during nonhomologous end joining. Cell Rep 20(12):2810–2819. https://doi.org/10.1016/j.celrep.2017.08.091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hammel M, Rey M, Yu Y, Mani RS, Classen S, Liu M, Pique ME, Fang S, Mahaney BL, Weinfeld M, Schriemer DC, Lees-Miller SP, Tainer JA (2011) XRCC4 protein interactions with XRCC4-like factor (XLF) create an extended grooved scaffold for DNA ligation and double strand break repair. J Biol Chem 286(37):32638–32650. https://doi.org/10.1074/jbc.M111.272641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mahaney BL, Hammel M, Meek K, Tainer JA, Lees-Miller SP (2013) XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair. Biochem Cell Biol 91(1):31–41. https://doi.org/10.1139/bcb-2012-0058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Williams GJ, Hammel M, Radhakrishnan SK, Ramsden D, Lees-Miller SP, Tainer JA (2014) Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time. DNA Repair (Amst) 17:110–120. https://doi.org/10.1016/j.dnarep.2014.02.009

    Article  CAS  Google Scholar 

  38. Zhao B, Watanabe G, Morten MJ, Reid DA, Rothenberg E, Lieber MR (2019) The essential elements for the noncovalent association of two DNA ends during NHEJ synapsis. Nat Commun 10(1):3588. https://doi.org/10.1038/s41467-019-11507-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Eckelmann BJ, Bacolla A, Wang H, Ye Z, Guerrero EN, Jiang W, El-Zein R, Hegde ML, Tomkinson AE, Tainer JA, Mitra S (2020) XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells. NAR. Cancer 2(3):zcaa013. https://doi.org/10.1093/narcan/zcaa013

    Article  Google Scholar 

  40. Hammel M, Rashid I, Sverzhinsky A, Pourfarjam Y, Tsai MS, Ellenberger T, Pascal JM, Kim IK, Tainer JA, Tomkinson AE (2021) An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA ligase IIIalpha within a flexible DNA repair complex. Nucleic Acids Res 49(1):306–321. https://doi.org/10.1093/nar/gkaa1188

    Article  CAS  PubMed  Google Scholar 

  41. Staresincic L, Fagbemi AF, Enzlin JH, Gourdin AM, Wijgers N, Dunand-Sauthier I, Giglia-Mari G, Clarkson SG, Vermeulen W, Scharer OD (2009) Coordination of dual incision and repair synthesis in human nucleotide excision repair. EMBO J 28(8):1111–1120. https://doi.org/10.1038/emboj.2009.49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Rambo RP, Tainer JA (2010) Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr Opin Struct Biol 20(1):128–137. https://doi.org/10.1016/j.sbi.2009.12.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rambo RP, Tainer JA (2013) Super-resolution in solution X-ray scattering and its applications to structural systems biology. Annu Rev Biophys 42:415–441. https://doi.org/10.1146/annurev-biophys-083012-130301

    Article  CAS  PubMed  Google Scholar 

  44. Putnam CD, Hammel M, Hura GL, Tainer JA (2007) X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys 40(3):191–285. https://doi.org/10.1017/S0033583507004635

    Article  CAS  PubMed  Google Scholar 

  45. Brosey CA, Tainer JA (2019) Evolving SAXS versatility: solution X-ray scattering for macromolecular architecture, functional landscapes, and integrative structural biology. Curr Opin Struct Biol 58:197–213. https://doi.org/10.1016/j.sbi.2019.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Brosey CA, Ho C, Long WZ, Singh S, Burnett K, Hura GL, Nix JC, Bowman GR, Ellenberger T, Tainer JA (2016) Defining NADH-driven Allostery regulating apoptosis-inducing factor. Structure 24(12):2067–2079. https://doi.org/10.1016/j.str.2016.09.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hura GL, Menon AL, Hammel M, Rambo RP, Poole FL 2nd, Tsutakawa SE, Jenney FE Jr, Classen S, Frankel KA, Hopkins RC, Yang SJ, Scott JW, Dillard BD, Adams MW, Tainer JA (2009) Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nat Methods 6(8):606–612. https://doi.org/10.1038/nmeth.1353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Classen S, Rodic I, Holton J, Hura GL, Hammel M, Tainer JA (2010) Software for the high-throughput collection of SAXS data using an enhanced Blu-ice/DCS control system. J Synchrotron Radiat 17(6):774–781. https://doi.org/10.1107/S0909049510028566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Classen S, Hura GL, Holton JM, Rambo RP, Rodic I, McGuire PJ, Dyer K, Hammel M, Meigs G, Frankel KA, Tainer JA (2013) Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the advanced light source. J Appl Crystallogr 46(Pt 1):1–13. https://doi.org/10.1107/S0021889812048698

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Dyer KN, Hammel M, Rambo RP, Tsutakawa SE, Rodic I, Classen S, Tainer JA, Hura GL (2014) High-throughput SAXS for the characterization of biomolecules in solution: a practical approach. Methods Mol Biol 1091:245–258. https://doi.org/10.1007/978-1-62703-691-7_18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Hura GL, Tsai CL, Claridge SA, Mendillo ML, Smith JM, Williams GJ, Mastroianni AJ, Alivisatos AP, Putnam CD, Kolodner RD, Tainer JA (2013) DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals. Proc Natl Acad Sci U S A 110(43):17308–17313. https://doi.org/10.1073/pnas.1308595110

    Article  PubMed  PubMed Central  Google Scholar 

  52. Rosenberg DJ, Syed A, Tainer JA, Hura GL Monitoring nuclease activity by X-ray scattering interferometry using gold nanoparticle conjugated DNA. Methods Mol Biol. (In this issue)

    Google Scholar 

  53. Mertens HD, Svergun DI (2010) Structural characterization of proteins and complexes using small-angle X-ray solution scattering. J Struct Biol 172(1):128–141. https://doi.org/10.1016/j.jsb.2010.06.012

    Article  CAS  PubMed  Google Scholar 

  54. Tang HYH, Tainer JA, Hura GL (2017) High resolution distance distributions determined by X-ray and Neutron scattering. Adv Exp Med Biol 1009:167–181. https://doi.org/10.1007/978-981-10-6038-0_10

    Article  CAS  PubMed  Google Scholar 

  55. Tsutakawa SE, Hura GL, Frankel KA, Cooper PK, Tainer JA (2007) Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography. J Struct Biol 158(2):214–223. https://doi.org/10.1016/j.jsb.2006.09.008

    Article  CAS  PubMed  Google Scholar 

  56. Hammel M, Yu Y, Fang S, Lees-Miller SP, Tainer JA (2010) XLF regulates filament architecture of the XRCC4.Ligase IV complex. Structure 18(11):1431–1442. https://doi.org/10.1016/j.str.2010.09.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Hammel M, Yu Y, Mahaney BL, Cai B, Ye R, Phipps BM, Rambo RP, Hura GL, Pelikan M, So S, Abolfath RM, Chen DJ, Lees-Miller SP, Tainer JA (2010) Ku and DNA-dependent protein kinase dynamic conformations and assembly regulate DNA binding and the initial non-homologous end joining complex. J Biol Chem 285(2):1414–1423. https://doi.org/10.1074/jbc.M109.065615

    Article  PubMed  Google Scholar 

  58. Hammel M, Yu Y, Radhakrishnan SK, Chokshi C, Tsai MS, Matsumoto Y, Kuzdovich M, Remesh SG, Fang S, Tomkinson AE, Lees-Miller SP, Tainer JA (2016) An intrinsically disordered APLF links Ku, DNA-PKcs, and XRCC4-DNA ligase IV in an extended flexible non-homologous end joining complex. J Biol Chem 291(53):26987–27006. https://doi.org/10.1074/jbc.M116.751867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Guo HF, Tsai CL, Terajima M, Tan X, Banerjee P, Miller MD, Liu X, Yu J, Byemerwa J, Alvarado S, Kaoud TS, Dalby KN, Bota-Rabassedas N, Chen Y, Yamauchi M, Tainer JA, Phillips GN Jr, Kurie JM (2018) Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe(2+)-binding. Nat Commun 9(1):512. https://doi.org/10.1038/s41467-018-02859-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Walker RG, Kattamuri C, Goebel EJ, Zhang F, Hammel M, Tainer JA, Linhardt RJ, Thompson TB (2021) Heparin-mediated dimerization of follistatin. Exp Biol Med (Maywood) 246(4):467–482. https://doi.org/10.1177/1535370220966296

    Article  CAS  Google Scholar 

  61. Cotner-Gohara E, Kim IK, Hammel M, Tainer JA, Tomkinson AE, Ellenberger T (2010) Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states. Biochemistry 49(29):6165–6176. https://doi.org/10.1021/bi100503w

    Article  CAS  PubMed  Google Scholar 

  62. Tsutakawa SE, Yan C, Xu X, Weinacht CP, Freudenthal BD, Yang K, Zhuang Z, Washington MT, Tainer JA, Ivanov I (2015) Structurally distinct ubiquitin- and sumo-modified PCNA: implications for their distinct roles in the DNA damage response. Structure 23(4):724–733. https://doi.org/10.1016/j.str.2015.02.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Roberts VA, Freeman HC, Olson AJ, Tainer JA, Getzoff ED (1991) Electrostatic orientation of the electron-transfer complex between plastocyanin and cytochrome c. J Biol Chem 266(20):13431–13441

    Article  CAS  Google Scholar 

  64. Rambo RP, Tainer JA (2010) Improving small-angle X-ray scattering data for structural analyses of the RNA world. RNA 16(3):638–646. https://doi.org/10.1261/rna.1946310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Thapar R, Wang JL, Hammel M, Ye R, Liang K, Sun C, Hnizda A, Liang S, Maw SS, Lee L, Villarreal H, Forrester I, Fang S, Tsai MS, Blundell TL, Davis AJ, Lin C, Lees-Miller SP, Strick TR, Tainer JA (2021) Mechanism of efficient double-strand break repair by a long non-coding RNA. Nucleic Acids Res 49(2):1199–1200. https://doi.org/10.1093/nar/gkaa1233

    Article  CAS  PubMed  Google Scholar 

  66. Zhou Y, Millott R, Kim HJ, Peng S, Edwards RA, Skene-Arnold T, Hammel M, Lees-Miller SP, Tainer JA, Holmes CFB, Glover JNM (2019) Flexible tethering of ASPP proteins facilitates PP-1c catalysis. Structure 27:1485–1496.e4. https://doi.org/10.1016/j.str.2019.07.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Hopkins JB, Gillilan RE, Skou S (2017) BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis. J Appl Crystallogr 50(Pt 5):1545–1553. https://doi.org/10.1107/S1600576717011438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Tully MD, Tarbouriech N, Rambo RP, Hutin S (2021) Analysis of SEC-SAXS data via EFA deconvolution and scatter. J Vis Exp 167:10.3791/61578

    Google Scholar 

  69. Hammel M, Amlanjyoti D, Reyes FE, Chen JH, Parpana R, Tang HY, Larabell CA, Tainer JA, Adhya S (2016) HU multimerization shift controls nucleoid compaction. Sci Adv 2(7):e1600650. https://doi.org/10.1126/sciadv.1600650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Manalastas-Cantos K, Konarev PV, Hajizadeh NR, Kikhney AG, Petoukhov MV, Molodenskiy DS, Panjkovich A, Mertens HDT, Gruzinov A, Borges C, Jeffries CM, Svergun DI, Franke D (2021) ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis. J Appl Crystallogr 54(1):343–355. https://doi.org/10.1107/S1600576720013412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Rambo RP, Tainer JA (2013) Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496(7446):477–481. https://doi.org/10.1038/nature12070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Durand D, Vives C, Cannella D, Perez J, Pebay-Peyroula E, Vachette P, Fieschi F (2010) NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers. J Struct Biol 169(1):45–53. https://doi.org/10.1016/j.jsb.2009.08.009

    Article  CAS  PubMed  Google Scholar 

  73. Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503. https://doi.org/10.1107/S0021889892001663

    Article  CAS  Google Scholar 

  74. Svergun D, Barberato C, Koch MHJ (1995) CRYSOL– a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28(6):768–773. https://doi.org/10.1107/S0021889895007047

    Article  CAS  Google Scholar 

  75. Schneidman-Duhovny D, Kim SJ, Sali A (2012) Integrative structural modeling with small angle X-ray scattering profiles. BMC Struct Biol 12:17. https://doi.org/10.1186/1472-6807-12-17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Schneidman-Duhovny D, Hammel M, Tainer JA, Sali A (2016) FoXS, FoXSDock and MultiFoXS: single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles. Nucleic Acids Res 44(W1):W424–W429. https://doi.org/10.1093/nar/gkw389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Schneidman-Duhovny D, Hammel M, Tainer JA, Sali A (2013) Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophys J 105(4):962–974. https://doi.org/10.1016/j.bpj.2013.07.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Schneidman-Duhovny D, Hammel M, Sali A (2010) FoXS: a web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res 38(Web Server issue):W540–W544. https://doi.org/10.1093/nar/gkq461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Forster F, Webb B, Krukenberg KA, Tsuruta H, Agard DA, Sali A (2008) Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies. J Mol Biol 382(4):1089–1106. https://doi.org/10.1016/j.jmb.2008.07.074

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Yang Z, Lasker K, Schneidman-Duhovny D, Webb B, Huang CC, Pettersen EF, Goddard TD, Meng EC, Sali A, Ferrin TE (2012) UCSF Chimera, MODELLER, and IMP: an integrated modeling system. J Struct Biol 179(3):269–278. https://doi.org/10.1016/j.jsb.2011.09.006

    Article  CAS  PubMed  Google Scholar 

  81. Webb B, Sali A (2016) Comparative protein structure modeling using modeller. Curr Protoc Bioinformatics 54:5 6 1–5 6 37. https://doi.org/10.1002/cpbi.3

    Article  Google Scholar 

  82. Hura GL, Hodge CD, Rosenberg D, Guzenko D, Duarte JM, Monastyrskyy B, Grudinin S, Kryshtafovych A, Tainer JA, Fidelis K, Tsutakawa SE (2019) Small angle X-ray scattering-assisted protein structure prediction in CASP13 and emergence of solution structure differences. Proteins 87(12):1298–1314. https://doi.org/10.1002/prot.25827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Andrec M, Snyder DA, Zhou Z, Young J, Montelione GT, Levy RM (2007) A large data set comparison of protein structures determined by crystallography and NMR: statistical test for structural differences and the effect of crystal packing. Proteins 69(3):449–465. https://doi.org/10.1002/prot.21507

    Article  CAS  PubMed  Google Scholar 

  84. Callaway E (2020) It will change everything': DeepMind's AI makes gigantic leap in solving protein structures. Nature 588(7837):203–204. https://doi.org/10.1038/d41586-020-03348-4

    Article  CAS  PubMed  Google Scholar 

  85. Reyes FE, Schwartz CR, Tainer JA, Rambo RP (2014) Methods for using new conceptual tools and parameters to assess RNA structure by small-angle X-ray scattering. Methods Enzymol 549:235–263. https://doi.org/10.1016/B978-0-12-801122-5.00011-8

    Article  CAS  PubMed  Google Scholar 

  86. Moore P (1980) Small-angle scattering. Information content and error analysis. J Appl Crystallogr 13(2):168–175. https://doi.org/10.1107/S002188988001179X

    Article  CAS  Google Scholar 

  87. Hura GL, Budworth H, Dyer KN, Rambo RP, Hammel M, McMurray CT, Tainer JA (2013) Comprehensive macromolecular conformations mapped by quantitative SAXS analyses. Nat Methods 10(6):453–454. https://doi.org/10.1038/nmeth.2453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank long-time collaborators and colleagues in the SAXS field, including Dmitry Svergun, Michal Hammel, Robert P. Rambo, Andrej Sali, Dina Schneidman, Jesse Hopkins, and Daniel Rosenberg, for their many insights and contributions including useful programs for SAXS data collection and analysis. Work is supported by NCI P01 CA092584 (to S.E.T., G.H., J.A.T.), NIGMS R01GM110387 (to S.E.T.), R35 CA220430 (to J.A.T.), and 1R01GM137021 (To S.E.T. and G.H.). JAT effort is also supported by Cancer Prevention Research Institute of Texas (CPRIT) grant RP180813 and a Robert A Welch Chemistry Chair. The SIBYLS beamline’s efforts are supported by DOE-BER IDAT under contract DE-AC02-05CH11231.

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Authors and Affiliations

  1. Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

    Naga Babu Chinnam, Aleem Syed & John A. Tainer

  2. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Kathryn H. Burnett, Greg L. Hura, John A. Tainer & Susan E. Tsutakawa

  3. Chemistry and Biochemistry Department, University of California Santa Cruz, Santa Cruz, CA, USA

    Greg L. Hura

  4. Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

    John A. Tainer

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  1. Naga Babu Chinnam
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  2. Aleem Syed
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  6. Susan E. Tsutakawa
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Correspondence to Susan E. Tsutakawa .

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  1. Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA

    Nima Mosammaparast

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Chinnam, N.B., Syed, A., Burnett, K.H., Hura, G.L., Tainer, J.A., Tsutakawa, S.E. (2022). Universally Accessible Structural Data on Macromolecular Conformation, Assembly, and Dynamics by Small Angle X-Ray Scattering for DNA Repair Insights. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2063-2_4

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