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Membrane Protein Structure
Calculations from experimental solid-
state NMR data
1
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Recent Developments at the BTRC.
Simultaneous Assignment and Structure Refinement.
Tian et al. (2012) J Magn Reson 214:42.
Membrane protein structure determination:
human chemokine receptor CXCR1 in liposomes
Marassi et al. (2011) Methods 55:363.
Das et al. (2012) J Am Chem Soc 134:2047.
Park et al. (2012) Nature 491:779.
Improved CS prediction by conformational sampling.
Tian et al. (2012) J Biomol NMR 54:237.
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Protein structures in the PDB.
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INPUT
protein amino acid sequence
experimental restraints
(CS, DC, CSA, PRE, distances)
STARTING MODEL
coarse-grained Rosetta model
(all-atom implicit membrane potential)
REFINE
NMR restrained MD (XPLOR-NIH)
VALIDATE
against all experimental data
Membrane protein structure calculation from solid-state NMR.
human CXCR1 in liposomes
(PDB 2LNL)
Marassi et al. (2011) Methods 55:363.
Das et al. (2012) J Am Chem Soc 134:2047.
Park et al. (2012) Nature 491:779.
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Improve chemical shift prediction by conformational sampling.
• Predict structural models and select ensemble based on energy and RMSD.
• Predict CS for each model in structural ensemble (ShiftX, SPARTA+, …).
• Average CS over all structures.
• Compare to experimental CS.
• CS reflect time-average of conformational
states of the protein.
• Prediction accuracy is improved by
averaging over ensemble of structures
predicted from sequence (i.e. with Rosetta).
• Potential for guiding NMR resonance
assignment.
Bacterial MerFt (PDB 2LJ2)
Tian et al. (2012) J Biomol NMR 54:237.
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Method for Simultaneous Assignment & Structure Refinement.
• AssignFit module developed for XPLOR-NIH.
• AssignFit example scripts and data files released with XPLOR-NIH 2.29.
fd coat protein in planar bilayers (PDB 2MZT)
Tian et al. (2012) J Magn Reson 214:42.
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Detection and analysis of dynamics from orientation restraints
Teriete et al. (2007) Biochemistry 46:6774.
human FXYD1 in micelles. fd coat protein in phospholipid bilayers
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Effective Energy Function / Implicit Membrane Model
(EEFx / IMMx) for XPLOR-NIH.
Energy Terms for NMR-restrained structure calculations
Experimental restraints
CDIH + NOE + PRE + DC + CSA …
Database / empirical restraints
torsionDBPot + RAMA + Rgyr …
Molecular interaction terms
BOND + ANGL + IMPR + DIHE +
VDWREPEL standard NMR calculations
VDWLJ + ELEC + SLV EEFx
VDWLJ + ELEC + SLV + IMM EEFx / IMMx
ETOTAL
EEXPERIMENTAL
+
EKNOWLEDGE
+
ESYSTEM
Clore & Gronenborn (1989) Crit Rev Biochem Mol Biol 24:479.
Lazaridis & Karplus (1999) Proteins 35:133.
Lazaridis (1999) Proteins 52:176.
Tian et al. (2014) Submitted.
Release with XPLOR-NIH 2.36.
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EEFx Effective Energy Function sustains native protein structures.
• Unrestrained MD simulations (1 ns, 300 K, real atomic masses, XPLOR-NIH).
• EEFx outperforms vacuum, VDWLJ and VDWREPEL force fields.
Staph. protein A Z-domain
(PDB: 1Q2N)
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EEFx Effective Energy Function sustains native protein structures.
•EEFx improves structural accuracy.
•EEFx improves RDC cross validation.
•EEFx optimizes H bonds.
•EEFx optimizes packing.
• Unrestrained MD simulations (100 ps, 300 K, real atomic masses, XPLOR-NIH).
• 11 test proteins with varying sizes (60-260 residues) and varying αβ folds.
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EEFx Effective Energy Function recognizes native fold.
• EEFx and Rosetta funnel towards native structure.
• EEFx provides wider dynamic range for discrimination of folded states.
Structures predicted with Rosetta and scored by Rosetta or EEFx energy.
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Protein G, B1 domain (PDB: 3GB1)
EEFx Effective Energy Function improves NMR structures:
effect on calculations with sparse restraints.
• Calculate structures from extended templates with simulated annealing.
• Restrain with dihedral angles and limited NOE distances (short-range NOEs removed; long-
range NOEs randomly eliminated from full data set).
•EEFx directs calculation towards native fold.
•EEFx improves structural accuracy.
•EEFx improves structural precision.
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• Calculate structures from extended templates with simulated annealing.
• Restrain with dihedral angles and all available NOE distances.
• Cross validate with RDCs.
•EEFx maintains agreement with other experimental
and conformational energy terms.
•EEFx improves structural quality.
•EEFx compares well with Water-refine.
•EEFx is computationally efficient: calculations are only
2.5 times longer than with REPEL.
EEFx Effective Energy Function improves NMR structures:
effect on structural quality.
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Introducing the membrane environment:
IMMx Implicit Membrane Model
• Model membrane environment based on known profiles (X-ray and neutron diffraction).
• Distance-dependent electrostatic screening and solvation.
fd coat protein (PDB: 2MZT)