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Autophagy pp 17–56Cite as

Structural Studies of Autophagy-Related Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1880))

Abstract

Information about the structure and dynamics of proteins is crucial for understanding their physiological functions as well as for the development of strategies to modulate these activities. In this chapter we will describe the work packages required to determine the three-dimensional structures of proteins involved in autophagy by using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Further we will provide instructions how to perform a molecular dynamics (MD) simulation using GABARAP as example protein.

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Acknowledgments

The authors acknowledge funding by the Deutsche Forschungsgemeinschaft (SFB1208 and SFB974) and the Jürgen Manchot Stiftung.

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Authors and Affiliations

  1. Institute of Complex Systems ICS-6 (Structural Biochemistry), Forschungszentrum Jülich, Jülich, Germany

    Melanie Schwarten, Oliver H. Weiergräber, Dušan Petrović, Birgit Strodel & Dieter Willbold

  2. Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany

    Birgit Strodel

  3. Institut für Physikalische Biologie, Heinrich Heine University Düsseldorf, Düsseldorf, Germany

    Dieter Willbold

  4. Department of Chemistry, BMC, Uppsala University, Uppsala, Sweden

    Dušan Petrović

Authors
  1. Melanie Schwarten
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  2. Oliver H. Weiergräber
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  3. Dušan Petrović
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  4. Birgit Strodel
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  5. Dieter Willbold
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Corresponding author

Correspondence to Dieter Willbold .

Editor information

Editors and Affiliations

  1. Signalling Programme, Babraham Institute, Cambridge, UK

    Nicholas Ktistakis

  2. Signalling Programme, Babraham Institute, Cambridge, UK

    Oliver Florey

Appendix

Appendix

In the following, the five .mdp files needed for the MD simulation using GROMACS, which is explained in Subheading 2.3, are provided.

1.1 “ions.mdp” for Adding Ions

;; ions.mdp ; Run setup integrator    = steep emtol    = 1000 emstep    = 0.01 nsteps    = 2000 ; Neighbor search cutoff-scheme    = Verlet pbc    = xyz ; Electrostatics and vdW coulombtype    = PME pme-order    = 4 fourierspacing    = 0.1 rcoulomb    = 1.2 rvdw    = 1.2

1.2 “em.mdp” for Energy Minimization

;; em.mdp ; Run setup integrator    = steep emtol    = 500 emstep    = 0.001 nsteps    = 2000 nstxout    = 100 ; Neighbor searching cutoff-scheme    = Verlet nstlist    = 20 ns-type    = grid pbc    = xyz ; Electrostatics coulombtype    = PME pme-order    = 4 fourierspacing    = 0.1 rcoulomb    = 1.2 ; VdW rvdw    = 1.2

1.3 “nvt.mdp” for the Equilibration MD Run in the NVT Ensemble

;; nvt.mdp define    = -DPOSRES ; Run setup integrator    = md dt    = 0.002 nsteps    = 50000 ; Output control nstxout    = 5000 nstvout    = 5000 nstfout    = 5000 nstlog    = 500 nstenergy    = 500 ; Bonds constraints    = all-bonds constraint-algorithm    = LINCS lincs-order    = 4 lincs-iter    = 1 ; Neighbor searching cutoff-scheme    = Verlet nstlist    = 20 ns-type    = grid pbc    = xyz ; Electrostatics coulombtype    = PME pme-order    = 4 fourierspacing    = 0.1 rcoulomb    = 1.2 ; VdW rvdw    = 1.2 ; T coupling is on tcoupl    = v-rescale tc-grps    = Protein Non-Protein tau-t    = 0.1 0.1 ref-t    = 298 298 nsttcouple    = 10 ; P coupling is off pcoupl    = no ; Velocity generation gen-vel    = yes gen-temp    = 298 continuation    = no

1.4 “npt.mdp” for the Equilibration MD Run in the NPT Ensemble

;; npt.mdp define    = -DPOSRES ; Run setup integrator    = md dt    = 0.002 nsteps    = 100000 ; Output control nstxout    = 5000 nstvout    = 5000 nstfout    = 5000 nstlog    = 500 nstenergy    = 500 ; Bonds constraints    = all-bonds constraint-algorithm    = LINCS lincs-order    = 4 lincs-iter    = 1 ; Neighbor searching cutoff-scheme    = Verlet nstlist    = 20 ns-type    = grid pbc    = xyz ; Electrostatics coulombtype    = PME pme-order    = 4 fourierspacing    = 0.1 rcoulomb    = 1.2 ; VdW rvdw    = 1.2 ; T coupling is on tcoupl    = v-rescale tc-grps    = Protein Non-Protein tau-t    = 0.1 0.1 ref-t    = 298 298 nsttcouple    = 10 ; P coupling is on pcoupl    = berendsen pcoupltype    = isotropic tau_p    = 1.0 ref_p    = 1.0 compressibility    = 4.5e-5 refcoord_scaling    = com ; Velocity generation gen-vel    = no gen-temp    = 298 continuation    = yes

1.5 “md.mdp” for the Production MD Run

;; md.mdp ; Run setup integrator    = md dt    = 0.002 nsteps    = 5000000 ; Output control nstxout    = 0 nstvout    = 0 nstfout    = 0 nstlog    = 2500 nstenergy    = 2500 nstxout-compressed    = 2500 compressed-x-grps    = System ; Bonds constraints    = all-bonds constraint-algorithm    = LINCS lincs-order    = 4 lincs-iter    = 1 ; Neighbor searching cutoff-scheme    = Verlet nstlist    = 20 ns-type    = grid pbc    = xyz ; Electrostatics coulombtype    = PME pme-order    = 4 fourierspacing    = 0.1 rcoulomb    = 1.2 ; VdW rvdw    = 1.2 ; T coupling is on tcoupl    = v-rescale tc-grps    = Protein Non-Protein tau-t    = 0.1 0.1 ref-t    = 298 298 nsttcouple    = 10 ; Pressure coupling is off pcoupl    = Parrinello-Rahman pcoupltype    = isotropic tau_p    = 2.0 ref_p    = 1.0 compressibility    = 4.5e-5 ; Velocity generation gen-vel    = no gen-temp    = 298 continuation    = yes

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Schwarten, M., Weiergräber, O.H., Petrović, D., Strodel, B., Willbold, D. (2019). Structural Studies of Autophagy-Related Proteins. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_2

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  • DOI: https://doi.org/10.1007/978-1-4939-8873-0_2

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