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Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS).

Hura GL, Menon AL, Hammel M, Rambo RP, Poole FL, Tsutakawa SE, Jenney FE, Classen S, Frankel KA, Hopkins RC, Yang SJ, Scott JW, Dillard BD, Adams MW, Tainer JA - Nat. Methods (2009)

Bottom Line: We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS).We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution.SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins.

View Article: PubMed Central - PubMed

Affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

ABSTRACT
We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

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SAXS provides accurate shape and assembly in solution for most samples. (a) For the ten proteins with structural homologs or existing structures, the experimental scattering data (colors) were compared with the scattering curve calculated for the matching structure (black). (b) For monodisperse samples, the envelope determinations (colored as in a) were overlaid with the existing structures (ribbons). All monomeric units had a seven amino-acid His-tag attached. (c) For the 9 proteins with no pre-existing structural information, envelope predictions from two independent programs were compared and generally agree. The DAMMIN results (black mesh) were generated without symmetry. The GASBOR results used 2-fold symmetry for PF0014/0015, PF0965/0966/0967/0971, PF1911 (dimer), PF00716 (dimer), PF0699 (dimer) and PF1950 (dimer). Four-fold symmetry was imposed on tetrameric PF1291 and PF1372. (d) Plotting the SAXS data as I*q2 vs. q (Kratky plot) highlights proteins with large unfolded regions. The Kratky plot of PF0715 is shown for comparison of a folded protein and shows characteristic parabolic behavior at wide angles. In contrast PF0706.1, PF2047.1, and PF1282/1205 have SAXS data consistent with unfolded regions as reflected in the non-parabolic wide-angle properties.
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Figure 3: SAXS provides accurate shape and assembly in solution for most samples. (a) For the ten proteins with structural homologs or existing structures, the experimental scattering data (colors) were compared with the scattering curve calculated for the matching structure (black). (b) For monodisperse samples, the envelope determinations (colored as in a) were overlaid with the existing structures (ribbons). All monomeric units had a seven amino-acid His-tag attached. (c) For the 9 proteins with no pre-existing structural information, envelope predictions from two independent programs were compared and generally agree. The DAMMIN results (black mesh) were generated without symmetry. The GASBOR results used 2-fold symmetry for PF0014/0015, PF0965/0966/0967/0971, PF1911 (dimer), PF00716 (dimer), PF0699 (dimer) and PF1950 (dimer). Four-fold symmetry was imposed on tetrameric PF1291 and PF1372. (d) Plotting the SAXS data as I*q2 vs. q (Kratky plot) highlights proteins with large unfolded regions. The Kratky plot of PF0715 is shown for comparison of a folded protein and shows characteristic parabolic behavior at wide angles. In contrast PF0706.1, PF2047.1, and PF1282/1205 have SAXS data consistent with unfolded regions as reflected in the non-parabolic wide-angle properties.

Mentions: Six samples had SAXS curves that matched those calculated from single multimeric states suggested by PDB structures (Fig. 3b). PF1281 was initially aggregated based upon the SAXS results, but a homogenous solution was obtained after spin column filtration just prior to data collection (Fig. 2b).


Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS).

Hura GL, Menon AL, Hammel M, Rambo RP, Poole FL, Tsutakawa SE, Jenney FE, Classen S, Frankel KA, Hopkins RC, Yang SJ, Scott JW, Dillard BD, Adams MW, Tainer JA - Nat. Methods (2009)

SAXS provides accurate shape and assembly in solution for most samples. (a) For the ten proteins with structural homologs or existing structures, the experimental scattering data (colors) were compared with the scattering curve calculated for the matching structure (black). (b) For monodisperse samples, the envelope determinations (colored as in a) were overlaid with the existing structures (ribbons). All monomeric units had a seven amino-acid His-tag attached. (c) For the 9 proteins with no pre-existing structural information, envelope predictions from two independent programs were compared and generally agree. The DAMMIN results (black mesh) were generated without symmetry. The GASBOR results used 2-fold symmetry for PF0014/0015, PF0965/0966/0967/0971, PF1911 (dimer), PF00716 (dimer), PF0699 (dimer) and PF1950 (dimer). Four-fold symmetry was imposed on tetrameric PF1291 and PF1372. (d) Plotting the SAXS data as I*q2 vs. q (Kratky plot) highlights proteins with large unfolded regions. The Kratky plot of PF0715 is shown for comparison of a folded protein and shows characteristic parabolic behavior at wide angles. In contrast PF0706.1, PF2047.1, and PF1282/1205 have SAXS data consistent with unfolded regions as reflected in the non-parabolic wide-angle properties.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094553&req=5

Figure 3: SAXS provides accurate shape and assembly in solution for most samples. (a) For the ten proteins with structural homologs or existing structures, the experimental scattering data (colors) were compared with the scattering curve calculated for the matching structure (black). (b) For monodisperse samples, the envelope determinations (colored as in a) were overlaid with the existing structures (ribbons). All monomeric units had a seven amino-acid His-tag attached. (c) For the 9 proteins with no pre-existing structural information, envelope predictions from two independent programs were compared and generally agree. The DAMMIN results (black mesh) were generated without symmetry. The GASBOR results used 2-fold symmetry for PF0014/0015, PF0965/0966/0967/0971, PF1911 (dimer), PF00716 (dimer), PF0699 (dimer) and PF1950 (dimer). Four-fold symmetry was imposed on tetrameric PF1291 and PF1372. (d) Plotting the SAXS data as I*q2 vs. q (Kratky plot) highlights proteins with large unfolded regions. The Kratky plot of PF0715 is shown for comparison of a folded protein and shows characteristic parabolic behavior at wide angles. In contrast PF0706.1, PF2047.1, and PF1282/1205 have SAXS data consistent with unfolded regions as reflected in the non-parabolic wide-angle properties.
Mentions: Six samples had SAXS curves that matched those calculated from single multimeric states suggested by PDB structures (Fig. 3b). PF1281 was initially aggregated based upon the SAXS results, but a homogenous solution was obtained after spin column filtration just prior to data collection (Fig. 2b).

Bottom Line: We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS).We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution.SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins.

View Article: PubMed Central - PubMed

Affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

ABSTRACT
We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

Show MeSH