Results tagged “SAXS” from The SIBYLS Beamline

We bring to your attention a nice review published recently in the European Biophysics Journal describing the theoretical and practical considerations when using SAXS to characterize macromolecular flexibility.


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The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.


SAXS was used to characterize the structural effects of phosphorylation events that modulate the ability of retinoblastoma protein to associate with E2F and other proteins. This work has been published in Genes & Development in the May 8th Advanced Online Articles section.


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Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible ”pocket” domain loop such that it mimics and directly blocks E2F transactivation domain (E2FTD) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2FTD-pocket association and protein binding to the pocket ”LxCxE” site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.


Burke, J.R., Hura, G.L., and Rubin, S.M. “Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control.” Genes Dev. (May 2012)

Tsutakawa et al. have combined solution Small Angle X-ray Scattering (SAXS) data collected at the SIBYLS beamline with computational modeling carried out at Oak Ridge Leadership Computing Facility ( OLCF ) to elucidate new modes of flexibility in a key protein complex (Ubiquitin-PCNA) involved in DNA replication and repair. The work was published in the Oct 25th issue of PNAS, and has been highlighted by OLCF and the Faculty of 1000.

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PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)-a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations

Tsutakawa SE, Van Wynsberghe AW, Freudenthal BD, Weinacht CP, Gakhar L, Washington MT, Zhuang Z, Tainer JA, Ivanov I. “Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA.” Proc Natl Acad Sci U S A. 2011 Oct 17.

SAXS data collected at the SIBYLS beamline was used in conjunction with high resolution crystals structures to discern details of the unique interaction mode of of these key players in the autophagy pathway.


Mol_Cell_Nov4_2011.jpg

Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7NTD) recruits a unique “flexible region” from Atg3 (Atg3FR). The structure of an Atg7NTD-Atg3FR complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7∼Atg8-Atg3)2 complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1∼UBL-E2 architecture for enzymes mediating autophagy.

Taherbhoy AM, Tait SW, Kaiser SE, Williams AH, Deng A, Nourse A, Hammel M, Kurinov I, Rock CO, Green DR, Schulman BA. “Atg8 transfer from atg7 to atg3: a distinctive e1-e2 architecture and mechanism in the autophagy pathway.” Mol Cell 2011 Nov.;44(3):451-461.

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DNA double strand break repair via nonhomologous end joining is a critical regulatory function that maintains genomic integrity. One of the major factors involved in this process is the XLF-XRCC4 protein complex. Although mutation of either XLF or XRCC4 leads to defects in break repair, the function of the XLF-XRCC4 complex has remained enigmatic. In their Paper of the Week, Hammel et al. used structure-based methods to elucidate the mechanism by which XLF-XRCC4 promotes double strand break repair. The authors solved the crystal structure of the XLF-XRCC4 complex using the N-terminal head domains of each protein and identified two key structural features that stabilize the complex: a key-lock interaction that links the two proteins and a set of hydrogen-bonding interactions that supplement the key-lock bond. Furthermore, the authors found that the C-terminal domain of XLF was crucial for promoting the formation and extension of filaments of the XLF-XRCC4 complex, allowing for interaction with DNA in a concentration-dependent manner. The crystal structure also identified a putative DNA-binding region, located at the XLF-XRCC4 interface, which was confirmed through addition of DNA oligomers. Subsequent addition of the break repair complex nucleator Ku and DNA ligase IV allowed the authors to develop a model for nonhomologous end joining in which Ku initially binds the damaged DNA site and recruits the XLF-XRCC4 complex, which is necessary for proper alignment of damaged DNA for repair by DNA ligase IV. Importantly, the elucidation of the structure of the XLF-XRCC4 scaffold provides potential targets for anticancer therapeutics.

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. “XRCC4 protein interactions with XRCC4-like factor (XLF) create an extended grooved scaffold for DNA ligation and double strand break repair.” J Biol Chem. 2011 Sep 16;286(37):32638-50. Epub 2011 Jul 20.

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Unstructured proteins, RNA or DNA components provide functionally important flexibility that is key to many macromolecular assemblies throughout cell biology. As objective, quantitative experimental measures of flexibility and disorder in solution are limited, small angle scattering (SAS), and in particular small angle X-ray scattering (SAXS), provides a critical technology to assess macromolecular flexibility as well as shape and assembly. In a recent paper published in Biopolymers, Rob Rambo and John Tainer consider the Porod-Debye law as a powerful tool for detecting biopolymer flexibility in SAS experiments. They show that the Porod-Debye region fundamentally describes the nature of the scattering intensity decay, which captures information needed for distinguishing between folded and flexible particles. Particularly for comparative SAS experiments, application of the law, as described in their manuscript, can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition and interaction networks. This approach aids insightful analyses of fully and partly flexible macromolecules that is more robust and conclusive than traditional Kratky analyses. Furthermore, they demonstrate for prototypic SAXS data that the ability to calculate particle density by the Porod-Debye criteria provides an objective quality assurance parameter that may prove of general use for SAXS modeling and validation.

The figure, excerpted from the manuscript, shows SAXS experiments performed on an exemplary intrinsically disordered domain Rad51 AP1. Data was collected for both rad51 AP1 (red) and a fusion construct with E. coli maltose binding protein (MBP) (black). (A) Kratky plot demonstrating the parabolic shape for a partially folded particle (black) and hyperbolic shape for a full unfolded particle (red). (B) Porod-Debye plot demonstrating a clear plateau for the partially folded MBP-Rad51 AP1 hybrid particle. In the absence of MBP, the fully unfolded domain is devoid of any discernible plateau.

FoXS, a new webapp which uses the Debye formula for calculating theoretical scattering profiles, has been made available on the ModBase website. Give it a try to see how it compares to CRYSOL.

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FoXS is a method for computing a theoretical scattering profile of a structure and fitting of experimental profile. FoXS can be used as a basic tool for numerous structural modeling applications with SAXS data:

  • Comparison of solution and X-ray structure conformations
  • Modeling of different conformation, i.e. model active conformation starting from non-active
  • Structural characterization of flexible proteins
  • Assembly of multi domain proteins starting from single domain structures
  • Assembly of multi protein complexes
  • Fold recognition and comparative modeling
  • Determination of biologically relevant state from the crystals

Recent SAXS structures

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SAXS capabilities of the SIBYLS beamline are demonstrated quite nicely in recent papers published in September and October.

Datta AB, Hura GL, Wolberger C. “The structure and conformation of Lys63-linked tetraubiquitin.” J Mol Biol. 2009 Oct 9;392(5):1117-24. Epub 2009 Aug 4.
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Jain R, Hammel M, Johnson RE, Prakash L, Prakash S, Aggarwal AK. “Structural Insights into Yeast DNA Polymerase delta by Small Angle X-ray Scattering (SAXS).” J Mol Biol. 2009 Oct 7.
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Greenstein AE, Hammel M, Cavazos A, Alber T. “Interdomain communication in the Mycobacterium tuberculosis environmental phosphatase Rv1364c.” J Biol Chem. 2009 Aug 20.
link out

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A paper has been published in The October 2 issue of Cell by Scott Williams et al. that sheds light on a previously missing piece of the double-strand break repair complex MRN (aka Mre11-Rad50-Nbs1). The paper entitled “Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair” presents a compelling model of the role of Nbs1 (i.e “N” of the MRN) in coordinating double-strand break processing and repair. The paper was made possible in part by crystal structures and SAXS data of Nbs1 that were collected at the SIBYLS beamline.

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Abstract:

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a divalent MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

Williams RS, Dodson GE, Limbo O, Yamada Y, Williams JS, Guenther G, Classen S, Glover MJN, Iwasaki H, Russell P, Tainer JA. “Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair” Cell, Volume 139, Issue 1, 87-99, 2 October 2009
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In this paper, published in Nucleic Acid Research, the authors used small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of PNK. Extensive use of the SAXS capabilities of the SIBYLS beamline allowed the authors to visualize a flexible attachment of the FHA domain to the catalytic segment and localize the DNA in the DNA/PNK complex.

Bernstein NK, Hammel M, Mani RS, Weinfeld M, Pelikan M, Tainer JA, Glover JN. "Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase." Nucleic Acids Res. 2009 Aug 11. [epub].link out



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In a recent article in the General Physiology and Biophysics we describe an analysis tool using relatively inexpensive small angle X-ray scattering (SAXS) measurements to identify protein flexibility and validate a constructed minimal ensemble of models, which represent highly populated conformations in solution. The resolution of these results is sufficient to address the questions being asked: what kinds of conformations do the domains sample in solution? In our rigid body modeling strategy BILBOMD molecular dynamics (MD) simulations are used to explore conformational space. A common strategy is to perform the MD simulation on the domains connections at very high temperature, where the additional kinetic energy prevents the molecule from becoming trapped in a local minimum. The MD simulations provide an ensemble of molecular models from which a SAXS curve is calculated and compared to the experimental curve. A genetic algorithm is used to identify the minimal ensemble (minimal ensemble search, MES ) required to best fit the experimental data.

The SIBYLS beamline has recently been awarded 50,000 hours on the NERSC (National Energy Research Scientific Computing Center) to perform solution structure modeling using experimental SAXS data. Besides the usual ab-initio reconstructions programs a new approach in rigid body modeling BILBOMD has been parallelized on the NERSC supercomputer. It is commonly acknowledged that flexibility between domains of proteins is often critical for function. These motions, and proteins with large scale flexibility in general, are often not readily amenable to conventional structural analysis such as X-ray crystallography, NMR, or electron microscopy. We have developed an analysis tool using experimental SAXS measurements to identify flexibility and validate a constructed minimal ensemble of models which represent highly populated conformations in solution. The resolution is sufficient to address questions about the extent of the domain conformational sampling in solution? In our rigid body modeling strategy BILBOMD, molecular dynamics (MD) simulations are used to explore conformational space. A typical experiment involves  MD simulation on the domain connections at very high temperature, where the additional kinetic energy prevents the molecule from becoming trapped in a local minimum. The MD simulations provide an ensemble of molecular models from which a SAXS curve is calculated and compared to the experimental curve. A genetic algorithm is then used to identify the minimal ensemble (Minimal Ensemble Search, MES) required to best fit the experimental data. If you are interested in learning about and/or using this valuable SAXS analysis tool please contact Michal Hammel (MHammel at lbl dot gov).
In a recent article in the Journal of Molecular Biology a paper has been published exploring the ability of prokaryotic thermophiles to supply stable human protein homologs for structural biology. The authors have made use of an unusual deep-sea hydrothermal-vent worm called Alvinella pompejana. This worm has been found in temperatures averaging as high as 68 degrees C. The paper explores the structure, stability, and mechanism of Cu,Zn superoxide dismutase (SOD), an enzyme whose mutation is implicated in causing the neurodegenerative disease familial amyotrophic lateral sclerosis or Lou Gehrig's disease. The SAXS endstation of the SIBYLS beamline played a key role in providing confirmation of the dimeric state of the Alvinella pompejana SOD and its structural similarity to the Human SOD.

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In the September 19, 2008 issue of Cell, we report striking conformational rearrangements in the crystal structure of NEDD8~Cul5ctd-Rbx1 and SAXS analysis of NEDD8~Cul1ctd-Rbx1 relative to their unmodified counterparts. These results point to conformational control of Cullin-RING ligase (CRL) activity, with ligation of NEDD8 shifting equilibria to disfavor inactive closed architectures, and favor dynamic, open forms that promote polyubiquitination.

Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8~Cul5ctd-Rbx1 and SAXS analysis of NEDD8~Cul1ctd-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.

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Quen Cheng in the Cooper lab has done a nice set of experiments to address the usefulness of various readily available detergents for improving the behavior proteins in SAXS experiments.

BACK
Detergent  Category  Concentrations Shot  MW  CMC (mM)  CMC (%)  Type  Aggreg. # *  MW x N (kD) 
1-s-Nonyl-ß-D-thioglucoside 1 1x 322.4 2.9 0.093 N
C12E8 1 1x 539.1 0.1 0.006 N
C8E5 1 1x 350.5 7.1 0.249 N
CTAB 1 1x, 3x 364.5 1.0 0.036 I
DDAO 1 1X, 3x 201.4 10.4 0.209 N
DDMAB 1 0.5x, 1x, 2x 299.5 4.3 0.129 Z
FOS-Choline®-10 1 1x 323.4 13.0 0.420 Z
FOS-Choline®-12 1 1x 315.5 1.5 0.047 Z
FOS-Choline®-9 1 1x 309.4 19.0 0.588 Z
n-Decanoylsucrose 1 1x 496.6 2.5 0.124 N 55 27.31
n-Decyl-ß-D-thiomaltoside 1 1x 498.6 0.9 0.045 N 41 20.44
n-Dodecyl-N,N-dimethylglycine 1 1x 271.4 1.5 0.041 Z 76 20.63
n-Dodecyl-ß-D-maltoside 1 1x 510.6 0.2 0.009 N 100-155 65.10
n-Hexadecyl-ß-D-maltoside 1 0.5x, 1x, 2x 566.6 0.0006 0.000 N
n-Nonyl-ß-D-maltoside 1 1x 484.6 3.2 0.155 N
NP-40 1 1x, 2x 615.0 0.1 0.007
n-Tetradecyl-ß-D-maltoside 1 1x 538.6 0.01 0.001 N 120-127 66.52
n-Tridecyl-ß-D-maltoside 1 1x 524.6 0.033 0.002 N 98 51.41
n-Undecyl-ß-D-maltoside 1 1x 496.6 0.59 0.029 N 8.0-16 5.96
Zwittergent® 3-14 1 1x 363.6 0.4 0.015 Z
1-s-Heptyl-ß-D-thioglucoside 2 1x 294.4 29.0 0.854 N
1-s-Octyl-ß-D-thioglucoside 2 1x 308.4 9.0 0.278 N
C12E9 2 1x 583.1 0.1 0.005 N
C-HEGA-11 2 1x 391.5 11.5 0.450 N
C-HEGA-8 2 0.5x, 1x, 2x 293.3 180.0 5.279 N
Cymal®-1 2 1x 438.5 340.0 14.909 N
Cymal®-5 2 1x 494.5 2.4 0.119 N
Cymal®-6 2 1x 508.5 0.6 0.028 N
HECAMEG 2 1x 335.4 19.5 0.654 N
HEGA-10 2 1x 379.5 7.0 0.266 N
Heptyl-ß-D-thioglucoside 2 1x 274.3 30.0 0.823 N
IPTG 2 1x 238.3 None N 4.0-14 2.14
MEGA-9 2 1x 335.5 25.0 0.839 N
n-Decyl-ß-D-maltoside 2 1x 482.6 1.8 0.087 N
n-Nonyl-ß-D-maltoside 2 1x 468.4 6.0 0.281 N
n-Octyl-ß-D-glucoside 2 0.5x, 1x, 2x 292.4 24.5 0.716 N
Nonyl-ß-D-glucoside 2 0.5x, 1x, 2x 306.4 6.5 0.199 N
Sucrose monolaurate 2 1x 524.6 0.2 0.010 N 84 44.07
TRITON® X-100 2 0.5x, 1x, 2x 631.0 0.9 0.057 N
Zwittergent® 3-10 2 1x 307.6 40.0 1.230 Z
Zwittergent® 3-12 2 1x 335.6 4.0 0.134 Z
Zwittergent® 3-08 2 1x 279.6 330.0 9.227 Z
Anapoe® 20 3 1x 1227.5 0.059 0.007 N
Anapoe® 35 3 1x None None N
Anapoe® 56 3 1x None None N
Anapoe® 58 3 1x None None N
Anapoe® 80 3 1x 1309.7 0.012 0.002 N
Anapoe® C10E6 3 1x 427.1 0.9 0.038 N
Anapoe® C10E9 3 1x None None N
Anapoe® C12E10 3 1x None None N
Anapoe® C13E8 3 1x None None N
Anapoe® X-114 3 1x None None N
Anapoe® X-305 3 1x None None N
Anapoe® X-405 3 1x None None N
BAM 3 1x 384.5 None I
CHAPS 3 0.5x, 1x, 2x 614.9 8.0 0.492 Z
CHAPSO 3 1x 630.9 8.0 0.505 Z
C-HEGA-10 3 1x 377.5 35.0 1.321 N
C-HEGA-9 3 1x 363.5 108.0 3.926 N
Cymal®-2 3 1x 452.5 120.0 5.430 N
Cymal®-3 3 0.1x, 0.2x, 0.5x, 1x, 3x 466.5 34.5 1.609 N
Cymal®-4 3 1x 480.5 7.6 0.365 N
Deoxy BigChap 3 1x 862.1 1.4 0.121 N
FOS-Choline®-8 3 1x 295.4 102.0 3.013 Z
HEGA-8 3 1x 351.5 109.0 3.831 N
HEGA-9 3 1x 365.5 39.0 1.425 N
LDAO 3 0.5x, 1x 229.4 2.0 0.046 N
MEGA-8 3 0.1x, 1x, 3x 321.4 79.0 2.539 N
n-Hexyl-ß-D-glucoside 3 1x 264.3 250.0 6.608 N
n-Octanoylsucrose 3 1x 468.5 24.4 1.143 N 83 38.89
n-Octyl-ß-D-thiomaltoside 3 1x 470.6 9.0 0.424 N
Pluronic® F-68 3 1x ~8350 None N
Thesit® 3 1x 582.9 0.09 0.005 N

"category" - group designation based on 1xCMC scattering curve
"N" - non-ionic detergent
"I" - ionic detergent
"Z" - zwitterionic detergent
"Aggreg. #" - monomers per micelle (from CalBiochem)
"MW x N" - estimate of micelle molecular weight

Below CMC Above CMC
Group 1
(n=20)
Good for SAXS Possible with strong protein signal
Group 2
(n=22)
Good for SAXS Bad for SAXS
Group 3
(n=31)
Possible with strong protein signal Bad for SAXS

Here are the recommended concentrations of detergents that may contribute the least amount of background to SAXS studies. Omitted from this list are any detergents that would need to be used at concentrations < 0.1%, based on the above table and the detergent CMCs.

Common Detergents
0.1% CHAPS
0.2% DDMAB
0.5% OG

Group I less than 2xCMC
0.1% 1-s-Nonyl-β-D-thioglucoside
0.2% n-Decanoylsucrose
0.3% n-Nonyl-β-D-maltoside
0.4% DDAO
0.5% C8E5
0.8% FOS-Choline®-10
1.1% FOS-Choline®-9

Group II less than 1xCMC (rounded down)
0.1% Cymal®-5
0.1% Zwittergent®3-12
0.1% Nonyl-β-D-glucoside
0.2% HEGA-10
0.2% 1-s-Octyl-β-D-thioglucoside
0.2% n-Nonyl-β-D-maltoside (2)
0.4% C-HEGA-11
0.6% HECAMEG
0.8% Heptyl-β-D-thioglucoside
0.8% MEGA-9
0.8% 1-s-Heptyl-β-D-thioglucoside
1.2% Zwittergent® 3-10
5.2% C-HEGA-8
9.2% Zwittergent® 3-08
14.8% Cymal®-1

Group III less than 0.2xCMC
0.1% CHAPSO
0.2% n-Octanoylsucrose
0.3% C-HEGA-10
0.3% HEGA-9
0.3% Cymal®-3
0.5% MEGA-8
0.6% C-HEGA-9
1.0% Cymal®-2
1.2% n-Hexyl-β-D-glucoside

Detergents in SAXS

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Detergents can be extremely useful in preparing monodisperse samples for SAXS. However, they can also contribute enormous background scattering, potentially masking the desired protein signal entirely.

We recommend to not using detergents in entire protein preparation. We observed only a few cases (~0.1%) where the detergent ( bellow CMC concentration ) did not affect the protein signal. Basically, there is a high chance that detergent will make your SAXS data useless!!!!

It is crucial, therefore, to choose a detergent that will contribute the least amount of background scattering to your sample while improving sample monodispersity. The data presented here (see links at bottom of page) should be a useful starting point in choosing the right detergent for your sample. Use a detergent only in the case when the protein preparation without detergent was not successful

How we looked for good detergents

We searched for SAXS-compatible detergents by collecting data on 73 detergents in PBS. By subtracting the PBS signal from the detergent signal, we can observe the scattering contribution of detergent alone. We first collected data at 1xCMC (critical micelle concentration) to compare the scattering effects of different detergent types. Next, we investigated the effects of concentration relative to the CMC, collecting data ranging from 0.1xCMC to 3xCMC on a subset of the 73 detergents.

Observed Trends

Scattering signal is highly concentration-dependent (below CMC is much better than above CMC), but not all detergents are created equal. Based on the 1xCMC scattering curves, we derived three groups of detergents with scattering properties ranging from essentially no signal (group 1) to large protein-like signal (group 3). The following table provides a rough guide of detergent SAXS-compatibility:

Below CMC Above CMC
Group 1
(n=20)
Good for SAXS Possible with strong protein signal
Group 2
(n=22)
Good for SAXS Bad for SAXS
Group 3
(n=31)
Possible with strong protein signal Bad for SAXS

Caveats

  • 1xCMC may not be a very reliable data point
    • Sensitive to pipetting error
    • CMC fluctuates with temperature, pH, ionic strength
  • Majority of data from single experiment
  • Unclear why detergents scatter differently; no obvious trends in MW, micelle size, % concentration, etc.
  • Detergents alone may behave differently than detergents in complex with protein
  • Some level of subjectivity in "group" designations
  • Doesn't address membrane protein applications

What the data provides, however, is a starting point for finding a detergent that improves the monodispersity of your sample without dominating the SAXS signal.

Data

The following table include links to scattering curves of each concentration of detergent we analyzed, with Glucose Isomerase plotted as a reference protein curve.

A sortable Table of detergents.

List of best-bet detergents

Beamline Diagram

Focus Beam at Beamstop with Experimental Table Between PX and SAXS

Check Helium tank outside of hutch to make sure it is full and that it is flowing into the shutter box. It is important to purge all air from the shutterbox as the ion-guage needs to report a consistent value.

Turn Helium on as early as possible to assure shutterbox is purged

In a terminal window turn off the feedback with the following command:


feedback.com off

Switch KVM to the Beamline computer, and launch "Shortcut to BL Control Main".

Click the white arrow in upper left of main window. This will open the "Beamline 12.3.1 Beamline Control System" window.

Open bothe the Motor Debugger and Motor Monitor modules from the "Motors" pulldown menu.

Turn on autoscaling under the "Amplifiers" pulldown main menu. (why?)

Set M2 Bend Up to 244000, and click move button.

Set M2 Bend Down to 267000, and click move button.

Change "Mono eV" to 10000.

Remove the safety pin and use the hand crank to move the table to the mid point. There is a piece of red tape marked in pen "YAG" indicating the proper midpoint position of the table.

Attach the BNC cable to the camera that monitors the YAG prism, and focus the camera (this step will hopefully be unnecessary once a more permanent camera position is established.

Close the hutch.

Open the main shutter. You should see the direct beam hitting the YAG prism at this point.

Select "tune rocking curve" and click either the step ⬆ or the step ⬇ button once to initialize the optimization procedure. (this automagically adjusts Theta2)

Select "M2 Tilt" and click the home button to reset all values to 0.000

Move M2 Tilt to 12000.

Some PX users will have adjusted the Slits1 to make a very tight beam so they should checked and backed off if necessary. Aperture Line 1 and Aperture Line 11 values should be decreased several unit values.

If you lose the beam jog M2 Tilt in 100 unit increments (this should move the beam up and down on the video monitor).

Select "Chi2" (value should be ~0.492) jog Chi2 in 0.01 increments. (this should move the beam left and right on the video monitor). Because Chi2 is adjusting the focus of the beam changes to this value will drastically alter the shape of the beam.

Sometimes the table will need to be moved slightly in order to position the beam in the middle of the YAG. Additionally the shutter control cable may sometimes get in the way so it must be unplugged from the shutterbox. Turn off power on shutter control box and unplug cable.

Close hutch and turn off hutch light.

Open main shutter and make sure video monitor is turned on.

Optimize the shape and size of the beam. You want it as round and small as possible. Make small adjustments to both Chi2 and M2 Tilt.

Move Experimental Table to SAXS Position

Move the table all the way into SAXS mode using the hand crank. There is a digital dial attached to the air table's sub-frame. Crank the table until this value reads zero.

Re-attach the shutter control cable to the shutter box and turn on the power to the shutter control box. Press the small red button on the shutter control box to really turn it back on.

Close hutch and open main shutter.

Optimize Slits 1, Slits 2, and Slits3 - itertatively

Make sure that the video signal going to the beamline computer is displaying the Beam Position Monitor (BPM) video-feed from the camera that points into the shutter box.

Turn on feedback with the following command:

feedback.com on

The point here is to move slits1 (Aperture guys) and slits2 (SAXS aperture guys) and slits3 (Guard Slits guys) back in as far as possible without actually clipping the beam.

The beam on the back of the shutter may be a bit rough on the bottom edge as a result of nicking slits2 and may look like this:

Jog M2 Tilt to move the whole beam slightly up.

Make sure the High Voltage control unit that feeds the ion guage in the shutterbox has not been tripped. If it has been tripped then reset and increase voltage to as close to 300V as possible.

Close all four slits1 blades as far as possible without clipping beam.

  • Aperture Line 1 moves in from the right
  • Aperture Line 11 moves in from the left
  • Aperture Upper moves in from the top... duh
  • Aperture Lower moves in from the bottom... duh

Close all four slits2 blades as far as possible without clipping beam.

  • SAXS Aperture Line 1 moves in from the right
  • SAXS Aperture Line 11 moves in from the left
  • SAXS Aperture Upper moves in from the bottom... huh?
  • SAXS Aperture Lower moves in from the top... huh?

Insert all filters into direct beam and take a 1sec shot on the MAR CCD.

Close all four slits3 blades as far as possible without clipping beam.

  • Guard Slits 1 more + values to close blade down. (Left side)
  • Guard Slits 11 more - values to close blade down. (Right side)
  • Guard Slits Upper more + values to close blade down.
  • Guard Slits Lower more - values to close blade down.

Reduce Reflections from Slits3 and adjust beamstop

Take a 1 sec shot on MAR CCD. It will look something like this:

Back off the Guard Slits Upper and Guard Slits Lower until the vertical streaking is eliminated. (streaking toward the top of the screen is caused by Guard Slits Lower being too far in, and vice-a versa for streaking downwards)

Back off the Guard Slits 1 and Guard Slits 11 until the horizontal streaking is eliminated. (this is usually less of a problem, but again streaking to the left is caused by x-rays reflecting off of Guard Slits 11, and vice-a-versa for streaking to the right)

Adjust Endstop y to move the beamstop up and down.

Adjust Endstop x to move the beamstop left and right.

Center the beamstop by observing a line integration tool drawn though the beamstop. The idea is to get a symmetrical background scatter aboce and below the beamstop shadow.

Define Beam for Users

Enter hutch. Manually open the experimental shutter. Insert CCD shield. Insert sample cell with fluorescent paper.

Close hutch. Open main shutter and define the bounding box for the beam on the video monitor.

It may be necessary to move the sample up or down (Sample x and Sample y) so that the beam enters the sample cell in the center.

Enter hutch. Close experimental shutter. Remove CCD shield.

You are now ready to CRUSH!!!!!

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