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Structural studies of P-type ATPase-ligand complexes using an X-ray free-electron laser.

Bublitz M, Nass K, Drachmann ND, Markvardsen AJ, Gutmann MJ, Barends TR, Mattle D, Shoeman RL, Doak RB, Boutet S, Messerschmidt M, Seibert MM, Williams GJ, Foucar L, Reinhard L, Sitsel O, Gregersen JL, Clausen JD, Boesen T, Gotfryd K, Wang KT, Olesen C, Møller JV, Nissen P, Schlichting I - IUCrJ (2015)

Bottom Line: Consequently, membrane proteins are targeted by a large number of currently approved drugs.The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate.This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease - PUMPkin, Danish National Research Foundation, Aarhus University , Gustav Wieds Vej 10c, 8000 Aarhus C, Denmark.

ABSTRACT
Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein-ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

No MeSH data available.


Related in: MedlinePlus

Rwork and Rfree values resulting from different refinement strategies. (a) R values after refinement with increasing numbers of parameters, including data to 4.0 Å resolution. (b) Differences in R values after refinement (all atom coordinates, individual B factors) depending on the chosen high-resolution data cutoff. Models were refined in parallel to both the lower and the higher resolution cutoff and the R values were compared at the lower cutoff. The range of lower data quality between 3.6 and 3.4 Å is probably caused by high background scattering owing to the solvent.
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fig2: Rwork and Rfree values resulting from different refinement strategies. (a) R values after refinement with increasing numbers of parameters, including data to 4.0 Å resolution. (b) Differences in R values after refinement (all atom coordinates, individual B factors) depending on the chosen high-resolution data cutoff. Models were refined in parallel to both the lower and the higher resolution cutoff and the R values were compared at the lower cutoff. The range of lower data quality between 3.6 and 3.4 Å is probably caused by high background scattering owing to the solvent.

Mentions: Nevertheless, we obtained a clear solution by MR using the synchrotron structure (PDB entry 3n8g; Bublitz et al., 2013 ▸) with all ligands removed as a search model (see §1 for details; Fig. 1 ▸b). An initial rigid-body refinement with increasing numbers of rigid groups was carried out in PHENIX using data to 4 Å resolution, and finally an all-atom and individual B-factor refinement yielding a decrease in the model R factors (Fig. 2 ▸a). In order to determine the effective resolution limit of the data, we then carried out successive refinement runs with a stepwise inclusion of higher resolution shells, as suggested in recent work by Karplus & Diederichs (2012 ▸), Steuber et al. (2014 ▸) and Hattne et al. (2014 ▸) (Fig. 2 ▸b). This analysis revealed an improvement of model R factors upon the inclusion of higher resolution data, justifying an extension of the data set to 2.8 Å resolution. Only the resolution range between 3.6 and 3.4 Å led to increased R factors, most likely owing to high solvent background scattering in this resolution range. The strategy of expanded resolution led to a final refined model with Rwork and Rfree values of 30.6 and 34.4%, respectively, at 2.8 Å resolution.


Structural studies of P-type ATPase-ligand complexes using an X-ray free-electron laser.

Bublitz M, Nass K, Drachmann ND, Markvardsen AJ, Gutmann MJ, Barends TR, Mattle D, Shoeman RL, Doak RB, Boutet S, Messerschmidt M, Seibert MM, Williams GJ, Foucar L, Reinhard L, Sitsel O, Gregersen JL, Clausen JD, Boesen T, Gotfryd K, Wang KT, Olesen C, Møller JV, Nissen P, Schlichting I - IUCrJ (2015)

Rwork and Rfree values resulting from different refinement strategies. (a) R values after refinement with increasing numbers of parameters, including data to 4.0 Å resolution. (b) Differences in R values after refinement (all atom coordinates, individual B factors) depending on the chosen high-resolution data cutoff. Models were refined in parallel to both the lower and the higher resolution cutoff and the R values were compared at the lower cutoff. The range of lower data quality between 3.6 and 3.4 Å is probably caused by high background scattering owing to the solvent.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4491313&req=5

fig2: Rwork and Rfree values resulting from different refinement strategies. (a) R values after refinement with increasing numbers of parameters, including data to 4.0 Å resolution. (b) Differences in R values after refinement (all atom coordinates, individual B factors) depending on the chosen high-resolution data cutoff. Models were refined in parallel to both the lower and the higher resolution cutoff and the R values were compared at the lower cutoff. The range of lower data quality between 3.6 and 3.4 Å is probably caused by high background scattering owing to the solvent.
Mentions: Nevertheless, we obtained a clear solution by MR using the synchrotron structure (PDB entry 3n8g; Bublitz et al., 2013 ▸) with all ligands removed as a search model (see §1 for details; Fig. 1 ▸b). An initial rigid-body refinement with increasing numbers of rigid groups was carried out in PHENIX using data to 4 Å resolution, and finally an all-atom and individual B-factor refinement yielding a decrease in the model R factors (Fig. 2 ▸a). In order to determine the effective resolution limit of the data, we then carried out successive refinement runs with a stepwise inclusion of higher resolution shells, as suggested in recent work by Karplus & Diederichs (2012 ▸), Steuber et al. (2014 ▸) and Hattne et al. (2014 ▸) (Fig. 2 ▸b). This analysis revealed an improvement of model R factors upon the inclusion of higher resolution data, justifying an extension of the data set to 2.8 Å resolution. Only the resolution range between 3.6 and 3.4 Å led to increased R factors, most likely owing to high solvent background scattering in this resolution range. The strategy of expanded resolution led to a final refined model with Rwork and Rfree values of 30.6 and 34.4%, respectively, at 2.8 Å resolution.

Bottom Line: Consequently, membrane proteins are targeted by a large number of currently approved drugs.The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate.This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease - PUMPkin, Danish National Research Foundation, Aarhus University , Gustav Wieds Vej 10c, 8000 Aarhus C, Denmark.

ABSTRACT
Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein-ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

No MeSH data available.


Related in: MedlinePlus