Normal mode refinement of anisotropic thermal parameters for a supramolecular complex at 3.42-Å crystallographic resolution

Poon, Billy K.; Chen, Hong; Lu, Mingyang; Vyas, Nand K.; Quiocho, Florante A.; Wang, Qinghua; Ma, Jianpeng

doi:10.1073/pnas.0701204104

Cited by 53 publications

(52 citation statements)

References 42 publications

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“…To start, several parameters for normal-mode-based refinement, including the number of low-frequency modes and the value of stiffness, were optimized by running parallel refinements (14)(15)(16)(17). We found that 75 modes with stiffness of 30 yielded the lowest R factors.…”

Section: Resultsmentioning

confidence: 99%

“…To eliminate the slight differences in structural statistics because of the use of different structural refinement programs or different versions of them, we generally reminimize the structural models from PDB (14)(15)(16)(17). In this study, the Kv1.2 structure (PDB ID code 2A79) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This was achieved by using a normal-mode-based X-ray crystallographic refinement method that have been found to be effective in improving structural refinement in a number of biological systems (14)(15)(16)(17). In all previous applications, the structural improvement was reflected in building missing side chains, retracing main chains, adding sugar rings, and achieving better fit between the models and the diffraction data (14)(15)(16)(17). But in this study, the structural improvement on Kv1.2 was the addition of ∼37% molecular mass of the entire transmembrane portion of the channel.…”

Section: Discussionmentioning

confidence: 99%

“…The method models anisotropic B factors for all atoms by using only a small set of low-frequency normal modes (14,18,19). Consequently, the number of parameters used in structural refinement is drastically reduced, which allows a better data-toparameter ratio, and the structural flexibility is more accurately described than by using conventional isotropic B-factor refinement.…”

mentioning

confidence: 99%

“…A normal-mode-based X-ray crystallographic refinement method was recently developed by us and its efficiency in improving structural models was shown in a number of systems such as a supramolecular complex (14), a membrane-bound ion channel (15), a heavily glycosylated protein (16), and other systems (17). The method models anisotropic B factors for all atoms by using only a small set of low-frequency normal modes (14,18,19).…”

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confidence: 99%

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Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Chen

Wang²,

Ni³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Voltage-dependent potassium channels (Kv) are homotetramers composed of four voltage sensors and one pore domain. Because of high-level structural flexibility, the first mammalian Kv structure, Kv1.2 at 2.9 Å, has about 37% molecular mass of the transmembrane portion not resolved. In this study, by applying a novel normalmode-based X-ray crystallographic refinement method to the original diffraction data and structural model, we established the structure of full-length Kv1.2 in its native form. This structure offers mechanistic insights into voltage sensing. Particularly, it shows a hydrophobic layer of about 10 Å at the midpoint of the membrane bilayer, which is likely the molecular basis for the observed "focused electric field" of Kv1.2 between the internal and external solutions. This work also demonstrated the potential of the refinement method in bringing up large chunks of missing densities, thus beneficial to structural refinement of many difficult systems.anisotropic B factors | ion channel | normal-mode refinement | voltage gating V oltage-dependent ion channels sense change in the voltage across cell membrane and respond by allowing specific ions in/out with high selectivity and efficiency (1). Among them, voltage-dependent potassium channels (Kv) have been most extensively studied (2-6). In an action potential, the Kv channel allows intracellular potassium getting out of the cell to return the membrane potential to the polarized state, the failure of which could cause overexcitability of neuron cells, leading to neuronal diseases (7,8). The Kv channel is a homotetramer with four voltage sensors and one central pore domain (9, 10). In each subunit, four transmembrane helices (S1, S2, S3, and S4) make up a voltage-sensor domain, and two transmembrane helices (S5 and S6) contribute to the central pore domain. There are several highly conserved positively charged residues on the S4 helix, the so-called gating charges, which respond to voltage change to open or close the central pore domain (11).The first mammalian Kv crystal structure was of the Shaker family Kv1.2 from Rattus norvegicus [Protein Data Bank (PDB) ID code 2A79], determined to a resolution of 2.9 Å (10). Its structural model was refined by conventional isotropic X-ray crystallographic refinement protocol in CNS (12). Because of the high dynamic and/or static disordering, about 37% of the total molecular mass of the transmembrane portion (residues 131-421) was not resolved. For instance, all the loops connecting S1-S4 helices, and all the side chains on S1 and S3, as well as some of the side chains on S2 and S4, were missing (Fig. 1A). In order to obtain a clearer picture of Kv channels, the structure of a "paddle chimera" Kv was recently determined at a higher resolution of 2.4 Å (13). The improvements in the structure were achieved by crystallizing the channel in a lipid environment and by replacing the voltage-sensor paddle of Kv1.2 with the corresponding region of Kv2.1, a closely related Kv channel in the Shab family. Even though the ch...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Chen

Wang²,

Ni³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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222

220

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Protein Functional Motions: Basic Concepts and Computational Methodologies

Fuchigami

Fujisaki

Matsunaga

et al. 2011

Advancing Theory for Kinetics and Dynamics of Complex, Many‐Dimensional Systems: Clusters and Proteins

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Ensemble MD simulations restrained via crystallographic data: Accurate structure leads to accurate dynamics

Xue

Skrynnikov

2014

Protein Science

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Currently, the best existing molecular dynamics (MD) force fields cannot accurately reproduce the global free-energy minimum which realizes the experimental protein structure. As a result, long MD trajectories tend to drift away from the starting coordinates (e.g., crystallographic structures). To address this problem, we have devised a new simulation strategy aimed at protein crystals. An MD simulation of protein crystal is essentially an ensemble simulation involving multiple protein molecules in a crystal unit cell (or a block of unit cells). To ensure that average protein coordinates remain correct during the simulation, we introduced crystallography-based restraints into the MD protocol. Because these restraints are aimed at the ensemble-average structure, they have only minimal impact on conformational dynamics of the individual protein molecules. So long as the average structure remains reasonable, the proteins move in a native-like fashion as dictated by the original force field. To validate this approach, we have used the data from solid-state NMR spectroscopy, which is the orthogonal experimental technique uniquely sensitive to protein local dynamics. The new method has been tested on the well-established model protein, ubiquitin. The ensemble-restrained MD simulations produced lower crystallographic R factors than conventional simulations; they also led to more accurate predictions for crystallographic temperature factors, solid-state chemical shifts, and backbone order parameters. The predictions for 15 N R 1 relaxation rates are at least as accurate as those obtained from conventional simulations. Taken together, these results suggest that the presented trajectories may be among the most realistic protein MD simulations ever reported. In this context, the ensemble restraints based on high-resolution crystallographic data can be viewed as protein-specific empirical corrections to the standard force fields.

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Normal mode refinement of anisotropic thermal parameters for a supramolecular complex at 3.42-Å crystallographic resolution

Cited by 53 publications

References 42 publications

Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Protein Functional Motions: Basic Concepts and Computational Methodologies

Ensemble MD simulations restrained via crystallographic data: Accurate structure leads to accurate dynamics

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