Scott Classen: May 2012 Archives

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.


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.


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)

Poly ADP-ribosylation regulates cellular processes such as genomic stability maintenance, transcription and cell death. The structure of rat poly(ADP-ribose) glycohydrolase has been determined using crystallographic data collected at the SIBYLS beamline, giving insights into the enzyme’s endoglycosidase activity and providing a basis for the development of therapeutic inhibitors.


Reversible post-translational modification by poly(ADP-ribose) (PAR) regulates chromatin structure, DNA repair and cell fate in response to genotoxic stress. PAR glycohydrolase (PARG) removes PAR chains from poly ADP-ribosylated proteins to restore protein function and release oligo(ADP-ribose) chains to signal damage. Here we report crystal structures of mammalian PARG and its complex with a substrate mimic that reveal an open substrate-binding site and a unique ‘tyrosine clasp’ enabling endoglycosidic cleavage of branched PAR chains.

Kim, I.-K., Kiefer, J.R., Ho, C.M.W., Stegeman, R.A., Classen, S., Tainer, J.A., and Ellenberger, T. “Structure of mammalian poly(ADP-ribose) glycohydrolase reveals a flexible tyrosine clasp as a substrate-binding element.” Nat Struct Mol Biol. (May 2012)

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