3. As the non-sequential Kabat numbering scheme is used in the
crystal structures, we renumber the residues for convenience in
the simulation.
2.2 Simulation
Methods
2.2.1 All-Atom MD
Simulations
1. The systems were then solvated by TIP3P water molecules in
the cubic water box with minimal margin of 15 A ˚
´ from any
protein atom to any edge of water box, and sodium and chlorides were added to neutralize the system to a total concentration of ~150 mM.
2. The resulting solvated systems were energy minimized for
5000 conjugate gradient steps, with the protein fixed and
water molecules and counterions allowed to move, followed
by additional 5000 conjugate gradient steps, where all atoms
could move.
3. In the equilibration stage, each system was gradually relaxed by
performing a series of dynamic cycles, in which the harmonic
restraints on proteins were gradually removed to optimize the
protein-water interactions.
4. In the production stage, all simulations were performed using
the NPT ensemble at 310 K.
5. All MD simulations were performed using the NAMD software
[23] with CHARMM36 force field [24]. MD trajectories were
saved by every 2 ps for analysis.
6. To reduce the statistical noise, the systems of 1cu4, 1cu4 with
the inter-domain disulfide bond, 1cr9, and 1cr9 without the
inter-domain disulfide were repeated independently. The initial
structure of each individual repeat system was minimized by
using different energy minimization protocols. The systems
were then re-solvated randomly by water molecules and ions.
The initial velocity distribution of each repeat system was also
set differently. Thus, the repeat simulations were started from
alternate conformations.
2.2.2 Binding Energy
Evaluation
1. To evaluate the binding energy between Fab and the prion
peptide, the trajectory for each bound and apo system was
extracted from the last 20 ns of explicit solvent MD without
water molecules and ions.
2. The solvation energies of all systems were calculated using the
generalized Born method with molecular volume (GBMV)
after 500 steps of energy minimization to relax the local geometries caused by the thermal fluctuations which occurred in
the MD simulations.
3. In the GBMV calculation, the dielectric constant of water is set
to 80 and no distance cutoff is used. The binding energy
between two Fab and the prion peptide was calculated by
hE bind i ¼ hE complex i À hE Fab i À hE peptide i.
178
Jun Zhao et al.
crystal structures, we renumber the residues for convenience in
the simulation.
2.2 Simulation
Methods
2.2.1 All-Atom MD
Simulations
1. The systems were then solvated by TIP3P water molecules in
the cubic water box with minimal margin of 15 A ˚
´ from any
protein atom to any edge of water box, and sodium and chlorides were added to neutralize the system to a total concentration of ~150 mM.
2. The resulting solvated systems were energy minimized for
5000 conjugate gradient steps, with the protein fixed and
water molecules and counterions allowed to move, followed
by additional 5000 conjugate gradient steps, where all atoms
could move.
3. In the equilibration stage, each system was gradually relaxed by
performing a series of dynamic cycles, in which the harmonic
restraints on proteins were gradually removed to optimize the
protein-water interactions.
4. In the production stage, all simulations were performed using
the NPT ensemble at 310 K.
5. All MD simulations were performed using the NAMD software
[23] with CHARMM36 force field [24]. MD trajectories were
saved by every 2 ps for analysis.
6. To reduce the statistical noise, the systems of 1cu4, 1cu4 with
the inter-domain disulfide bond, 1cr9, and 1cr9 without the
inter-domain disulfide were repeated independently. The initial
structure of each individual repeat system was minimized by
using different energy minimization protocols. The systems
were then re-solvated randomly by water molecules and ions.
The initial velocity distribution of each repeat system was also
set differently. Thus, the repeat simulations were started from
alternate conformations.
2.2.2 Binding Energy
Evaluation
1. To evaluate the binding energy between Fab and the prion
peptide, the trajectory for each bound and apo system was
extracted from the last 20 ns of explicit solvent MD without
water molecules and ions.
2. The solvation energies of all systems were calculated using the
generalized Born method with molecular volume (GBMV)
after 500 steps of energy minimization to relax the local geometries caused by the thermal fluctuations which occurred in
the MD simulations.
3. In the GBMV calculation, the dielectric constant of water is set
to 80 and no distance cutoff is used. The binding energy
between two Fab and the prion peptide was calculated by
hE bind i ¼ hE complex i À hE Fab i À hE peptide i.
178
Jun Zhao et al.