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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: The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate.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.

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

Diffraction pattern, electron density and structure of SERCA–Ca2–AMPPCP. (a) Representative diffraction pattern of SERCA–Ca2–AMPPCP microcrystals. The boxed inset shows a strong Bragg spot at 2.8 Å resolution {Miller index [31, −15, 24], 〈I/σ(I)〉 = 8.4}. (b) Left, overall 2mFo − DFc electron-density map (contoured at 1.0σ) after final refinement including coordinates and B factors for all atoms and data to 2.8 Å resolution; right, molecular model, including ten transmembrane helices and cytoplasmic N (nucleotide binding), P (phosphorylation) and A (actuator) domains. The 13 structural segments initially refined as rigid groups are shown in different colours. Ligand-binding regions are indicated by numbers: 1, AMPPCP; 2, Ca2+ ions; 3, lipid.
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fig1: Diffraction pattern, electron density and structure of SERCA–Ca2–AMPPCP. (a) Representative diffraction pattern of SERCA–Ca2–AMPPCP microcrystals. The boxed inset shows a strong Bragg spot at 2.8 Å resolution {Miller index [31, −15, 24], 〈I/σ(I)〉 = 8.4}. (b) Left, overall 2mFo − DFc electron-density map (contoured at 1.0σ) after final refinement including coordinates and B factors for all atoms and data to 2.8 Å resolution; right, molecular model, including ten transmembrane helices and cytoplasmic N (nucleotide binding), P (phosphorylation) and A (actuator) domains. The 13 structural segments initially refined as rigid groups are shown in different colours. Ligand-binding regions are indicated by numbers: 1, AMPPCP; 2, Ca2+ ions; 3, lipid.

Mentions: SFX diffraction images of microcrystals of SERCA–Ca2–AMPPCP showed diffraction beyond 3 Å resolution (Fig. 1 ▸a). This is not on a par with the 2.5 Å resolution diffraction limit of the corresponding cryocooled macrocrystals at a synchrotron (Sørensen et al., 2004 ▸), and may be owing to a narrow time window for optimal microcrystal quality, in which an increase in the age of the crystal sample of only 24 h already led to weaker diffraction and a higher number of overlapping diffraction patterns, indicating crystal clustering. We recorded ∼760 000 images, of which ∼23 000 (3%) were identified as hits containing a diffraction pattern; of these, 4069 (∼18%) could be indexed in C2, the same space group as macroscopic SERCA–Ca2–AMPPCP crystals. The merged data have an overall ∼17-fold multiplicity of observation. This is extremely low for SFX data analysed by CrystFEL, which uses Monte Carlo methods to obtain scaled and merged intensities from the still images, averaging out all fluctuations in the data-acquisition process. One has to bear in mind, however, that in SFX multiplicity refers to the number of partial intensity measurements and not of fully integrated intensities. In line with this low multiplicity, the signal-to-noise ratio drops to 1.47 at 4 Å resolution (Table 1 ▸ and Supplementary Table S2). However, it must also be considered that the signal-to-noise ratio as defined by CrystFEL is based on the standard deviations of the sets of individual observations of a reflection themselves, not on the signal-to-noise ratios of merged, scaled and averaged diffraction peaks representing fully recorded reflections.


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)

Diffraction pattern, electron density and structure of SERCA–Ca2–AMPPCP. (a) Representative diffraction pattern of SERCA–Ca2–AMPPCP microcrystals. The boxed inset shows a strong Bragg spot at 2.8 Å resolution {Miller index [31, −15, 24], 〈I/σ(I)〉 = 8.4}. (b) Left, overall 2mFo − DFc electron-density map (contoured at 1.0σ) after final refinement including coordinates and B factors for all atoms and data to 2.8 Å resolution; right, molecular model, including ten transmembrane helices and cytoplasmic N (nucleotide binding), P (phosphorylation) and A (actuator) domains. The 13 structural segments initially refined as rigid groups are shown in different colours. Ligand-binding regions are indicated by numbers: 1, AMPPCP; 2, Ca2+ ions; 3, lipid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Diffraction pattern, electron density and structure of SERCA–Ca2–AMPPCP. (a) Representative diffraction pattern of SERCA–Ca2–AMPPCP microcrystals. The boxed inset shows a strong Bragg spot at 2.8 Å resolution {Miller index [31, −15, 24], 〈I/σ(I)〉 = 8.4}. (b) Left, overall 2mFo − DFc electron-density map (contoured at 1.0σ) after final refinement including coordinates and B factors for all atoms and data to 2.8 Å resolution; right, molecular model, including ten transmembrane helices and cytoplasmic N (nucleotide binding), P (phosphorylation) and A (actuator) domains. The 13 structural segments initially refined as rigid groups are shown in different colours. Ligand-binding regions are indicated by numbers: 1, AMPPCP; 2, Ca2+ ions; 3, lipid.
Mentions: SFX diffraction images of microcrystals of SERCA–Ca2–AMPPCP showed diffraction beyond 3 Å resolution (Fig. 1 ▸a). This is not on a par with the 2.5 Å resolution diffraction limit of the corresponding cryocooled macrocrystals at a synchrotron (Sørensen et al., 2004 ▸), and may be owing to a narrow time window for optimal microcrystal quality, in which an increase in the age of the crystal sample of only 24 h already led to weaker diffraction and a higher number of overlapping diffraction patterns, indicating crystal clustering. We recorded ∼760 000 images, of which ∼23 000 (3%) were identified as hits containing a diffraction pattern; of these, 4069 (∼18%) could be indexed in C2, the same space group as macroscopic SERCA–Ca2–AMPPCP crystals. The merged data have an overall ∼17-fold multiplicity of observation. This is extremely low for SFX data analysed by CrystFEL, which uses Monte Carlo methods to obtain scaled and merged intensities from the still images, averaging out all fluctuations in the data-acquisition process. One has to bear in mind, however, that in SFX multiplicity refers to the number of partial intensity measurements and not of fully integrated intensities. In line with this low multiplicity, the signal-to-noise ratio drops to 1.47 at 4 Å resolution (Table 1 ▸ and Supplementary Table S2). However, it must also be considered that the signal-to-noise ratio as defined by CrystFEL is based on the standard deviations of the sets of individual observations of a reflection themselves, not on the signal-to-noise ratios of merged, scaled and averaged diffraction peaks representing fully recorded reflections.

Bottom Line: The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate.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.

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