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Integrative Modeling of Macromolecular Assemblies from Low to Near-Atomic Resolution.

Xu X, Yan C, Wohlhueter R, Ivanov I - Comput Struct Biotechnol J (2015)

Bottom Line: By systematically combining various sources of structural, biochemical and biophysical information, integrative modeling approaches aim to provide a unified structural description of such assemblies, starting from high-resolution structures of the individual components and integrating all available information from low-resolution experimental methods.Second, we describe hybrid molecular dynamics, Rosetta Monte-Carlo and minimum ensemble search (MES) methods that can be used to incorporate SAXS into pseudoatomic structural models.We present concise descriptions of the two methods and their most popular alternatives, along with select illustrative applications to protein/nucleic acid assemblies involved in DNA replication and repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA.

ABSTRACT
While conventional high-resolution techniques in structural biology are challenged by the size and flexibility of many biological assemblies, recent advances in low-resolution techniques such as cryo-electron microscopy (cryo-EM) and small angle X-ray scattering (SAXS) have opened up new avenues to define the structures of such assemblies. By systematically combining various sources of structural, biochemical and biophysical information, integrative modeling approaches aim to provide a unified structural description of such assemblies, starting from high-resolution structures of the individual components and integrating all available information from low-resolution experimental methods. In this review, we describe integrative modeling approaches, which use complementary data from either cryo-EM or SAXS. Specifically, we focus on the popular molecular dynamics flexible fitting (MDFF) method, which has been widely used for flexible fitting into cryo-EM maps. Second, we describe hybrid molecular dynamics, Rosetta Monte-Carlo and minimum ensemble search (MES) methods that can be used to incorporate SAXS into pseudoatomic structural models. We present concise descriptions of the two methods and their most popular alternatives, along with select illustrative applications to protein/nucleic acid assemblies involved in DNA replication and repair.

No MeSH data available.


Related in: MedlinePlus

A MES ensemble of discrete Ub positions on PCNA best fit the experimental SAXS data for split-fusion (green) and cross-linked (blue) PCNAK164-Ub. (A) Schematic showing the MES methodology. (B) The scattering curve of the best MES ensemble fits the experimental scattering data better than the crystal structure 3L10.pdb. (C) P(r) plots. (D) Structures of the three models that as an ensemble best fit the experimental scattering curve are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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f0030: A MES ensemble of discrete Ub positions on PCNA best fit the experimental SAXS data for split-fusion (green) and cross-linked (blue) PCNAK164-Ub. (A) Schematic showing the MES methodology. (B) The scattering curve of the best MES ensemble fits the experimental scattering data better than the crystal structure 3L10.pdb. (C) P(r) plots. (D) Structures of the three models that as an ensemble best fit the experimental scattering curve are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mentions: Flexible positions of Ub on PCNA were then identified using BILBOMD [78] with the models from the MD simulation. This flexible position of Ub, along with the original MD positions, and the position observed in the crystal structure were then permuted on the homotrimer of PCNA to generate 130 PCNAK164-Ub PDB models, where each PCNA is modified by three Ubs. MES was then applied using this pool of models to fit either the split-fusion or the cross-linked PCNAK164-Ub SAXS profile. An ensemble of three models for each of the experimental constructs was identified, with the Ub being 25–30% in the crystallographic position, 40–50% in the computationally determined positions, and 25–30% flexible positions (Fig. 6). The result suggests a segmental flexibility of the Ub in PCNAK164-Ub, meaning that in solution Ub can adopt a number of discrete interchangeable positions on the surface of PCNA. This segmental flexibility of the Ub moiety on PCNA-Ub provides a variety of distinct positions capable of forming complexes with TLS polymerase, and, accordingly spatially organizes the PCNA-Ub interacting proteins for either efficient DNA replication or repair. These novel positions provided a rationalization for perplexing biochemical data e.g. explained the effects of mutations originally identified in genetic screens and known to interfere with TLS. The computationally derived positions, in an ensemble with the crystallographic position, provided the best fit to the solution scattering. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provided unexpected insight into the question of how Ub may help transition the TLS Pol from the back to the front side of PCNA to exchange with the replicative Pol [30].


Integrative Modeling of Macromolecular Assemblies from Low to Near-Atomic Resolution.

Xu X, Yan C, Wohlhueter R, Ivanov I - Comput Struct Biotechnol J (2015)

A MES ensemble of discrete Ub positions on PCNA best fit the experimental SAXS data for split-fusion (green) and cross-linked (blue) PCNAK164-Ub. (A) Schematic showing the MES methodology. (B) The scattering curve of the best MES ensemble fits the experimental scattering data better than the crystal structure 3L10.pdb. (C) P(r) plots. (D) Structures of the three models that as an ensemble best fit the experimental scattering curve are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588362&req=5

f0030: A MES ensemble of discrete Ub positions on PCNA best fit the experimental SAXS data for split-fusion (green) and cross-linked (blue) PCNAK164-Ub. (A) Schematic showing the MES methodology. (B) The scattering curve of the best MES ensemble fits the experimental scattering data better than the crystal structure 3L10.pdb. (C) P(r) plots. (D) Structures of the three models that as an ensemble best fit the experimental scattering curve are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mentions: Flexible positions of Ub on PCNA were then identified using BILBOMD [78] with the models from the MD simulation. This flexible position of Ub, along with the original MD positions, and the position observed in the crystal structure were then permuted on the homotrimer of PCNA to generate 130 PCNAK164-Ub PDB models, where each PCNA is modified by three Ubs. MES was then applied using this pool of models to fit either the split-fusion or the cross-linked PCNAK164-Ub SAXS profile. An ensemble of three models for each of the experimental constructs was identified, with the Ub being 25–30% in the crystallographic position, 40–50% in the computationally determined positions, and 25–30% flexible positions (Fig. 6). The result suggests a segmental flexibility of the Ub in PCNAK164-Ub, meaning that in solution Ub can adopt a number of discrete interchangeable positions on the surface of PCNA. This segmental flexibility of the Ub moiety on PCNA-Ub provides a variety of distinct positions capable of forming complexes with TLS polymerase, and, accordingly spatially organizes the PCNA-Ub interacting proteins for either efficient DNA replication or repair. These novel positions provided a rationalization for perplexing biochemical data e.g. explained the effects of mutations originally identified in genetic screens and known to interfere with TLS. The computationally derived positions, in an ensemble with the crystallographic position, provided the best fit to the solution scattering. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provided unexpected insight into the question of how Ub may help transition the TLS Pol from the back to the front side of PCNA to exchange with the replicative Pol [30].

Bottom Line: By systematically combining various sources of structural, biochemical and biophysical information, integrative modeling approaches aim to provide a unified structural description of such assemblies, starting from high-resolution structures of the individual components and integrating all available information from low-resolution experimental methods.Second, we describe hybrid molecular dynamics, Rosetta Monte-Carlo and minimum ensemble search (MES) methods that can be used to incorporate SAXS into pseudoatomic structural models.We present concise descriptions of the two methods and their most popular alternatives, along with select illustrative applications to protein/nucleic acid assemblies involved in DNA replication and repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA.

ABSTRACT
While conventional high-resolution techniques in structural biology are challenged by the size and flexibility of many biological assemblies, recent advances in low-resolution techniques such as cryo-electron microscopy (cryo-EM) and small angle X-ray scattering (SAXS) have opened up new avenues to define the structures of such assemblies. By systematically combining various sources of structural, biochemical and biophysical information, integrative modeling approaches aim to provide a unified structural description of such assemblies, starting from high-resolution structures of the individual components and integrating all available information from low-resolution experimental methods. In this review, we describe integrative modeling approaches, which use complementary data from either cryo-EM or SAXS. Specifically, we focus on the popular molecular dynamics flexible fitting (MDFF) method, which has been widely used for flexible fitting into cryo-EM maps. Second, we describe hybrid molecular dynamics, Rosetta Monte-Carlo and minimum ensemble search (MES) methods that can be used to incorporate SAXS into pseudoatomic structural models. We present concise descriptions of the two methods and their most popular alternatives, along with select illustrative applications to protein/nucleic acid assemblies involved in DNA replication and repair.

No MeSH data available.


Related in: MedlinePlus