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Molecular Computations A BBSI Tutorial

http://www.ccbb.pitt.edu/BBSI/index.htm

By:

Jeffry D. Madura Joshua A. Plumley Sankar Manepalli Thomas J. Dick

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Table of Contents

I.) QUANTUM CHEMISTRY CALCULATIONS USING GAUSSIAN03 AND GAUSSVIEW ............................... 3

HYDROGEN BOND STRENGTHS IN WATER, METHANOL, AND DIMETHYL ETHER

COMPLEXES ................................................................................................................... 3

CONFORMATIONAL ISOMERISM IN N-BUTANE ......................................................... 6

GLYCINE IN THE GAS PHASE AND IN WATER ............................................................ 9

II.) MOLECULAR DYNAMIC CALCULATIONS .............. 12

RAMACHANDRAN PLOT AND MOLECULAR DYNAMICS OF ALANINE DIPEPTIDE

(MOE) ........................................................................................................................ 12

DOCKING SIMULATIONS (MOE) .............................................................................. 15

III.) MOLECULAR DYNAMIC SIMULATIONS (NAMD) .... 17

SIMULATION OF BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI) IN VACUUM. 17

SIMULATION OF BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI) IN WATER ... 18

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I.) Quantum Chemistry Calculations using Gaussian03 and Gaussview

Hydrogen Bond Strengths in Water, Methanol, and Dimethyl Ether Complexes

For the quantum mechanical calculations we will calculate the actual binding energy

between two molecules. To do this, we will use the following equation:

( ) ( ) ( )E E AB E A E BΔ = − −

This shows the binding energy (ΔE) to be equal to the energy of the complex (the two

molecules together) minus the energies of the individual components (molecules A and B

in this case). We will compute the interaction energies with the Hartree-Fock (HF)

chemical method between a series of molecules (water, methanol, and dimethyl ether)

with the 3-21G basis set. These calculations will not take that long with this level of

theory (term used to incorporate the chemical method and basis set). Then all you have to

do is some simple math. All of the energy outputs will be in hartrees. However, the unit

that is more commonly utilized is kcal/mol. Therefore report binding energies in kcal/mol

employing the following conversion, 1 kcal/mol = 627.5095 hartrees.

To perform the calculations with Gaussian03, we will build the z-matrices in

GaussView (you can also do this manually, which allows a user to control molecular

symmetry much more vigorously). Open GaussView (referred to as GV below) and you

will see methane on one of the two screens that have opened.

Click on the tab that says Carbon Tetrahedral in the middle of the screen using the

left mouse button. This will open up the Select Element window. You can choose any

element you want by clicking on the periodic table and selecting an appropriate geometry

at the bottom of the screen. Click once with the left mouse button in the blue/purple

screen to generate a carbon tetrahedral (or the functional group shown in the main

window). To replace an atom on the generated carbon tetrahedral, click on the atom in

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the element selector window to make it “Hot”. Selecting an atom on the generated carbon

tetrahedral in the blue viewing window will be replaced by the “Hot” atom.

Example: When a carbon tetrahedral is shown in the main window and the carbon atom

is “Hot”, click once in the blue viewing window. A methane molecule should be

generated. Next, click on a hydrogen atom on the yielded methane. A ethane molecule

should be generated. One can easily see how a carbon chain may be created. Now lets try

something different. Click File → New → Create MolGroup. A new viewing window

will be opened. Next, create a methane in the new window. Now, make a hydrogen atom

“Hot” in the main window and click on a hydrogen atom on the methane in the viewing

window. See the difference from that described previously?

Tip: When two structures are within the same GV window the following shortcuts will be

useful. Place the cursor over the molecule you wish to alter. Second, press SHIFT +

ALT and the left mouse button. Lastly, move your cursor. Voila the molecule moves.

You should be able to move the corresponding structure without moving the other one. If

you wish to only rotate the structure without rotating the other one perform the previous

actions WITHOUT pressing SHIFT (in other words only press ALT).

To perform the quantum calculations, have the appropriate molecule in the

corresponding window selected. Select Calculate → Gaussian at the top of the main GV

window. In the Gaussian Calculation Setup window, click on Opt + Freq under Job type

tab; this will do a geometry optimization plus a frequency analysis. You can choose your

level of theory under the Method tab. You can also select a basis set by clicking on the

default basis set (3-21G) and changing it. Polarized and diffuse functions may be added

by the drop down menus within the next two columns next to the basis set (off to the

right). The default chemical method should be HF, which is what we will be using for

these optimizations. When you are ready to submit the calculation to Guassian, click

submit. Make a mental note of what directory the output file is saved too. Make a

mental note of what directory the output file is saved too. As the young and astute

students you are, you might be thinking to yourself, “Why is that bold and repeated

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twice?” “Did they make a typo?” Or maybe you are just thinking, “That must be

important,” which is of course the correct assumption.

After a computation is complete you can open up the output in GV by selecting

File → Open to observe the final optimized geometry. Other results may be observed as

well. Make sure the final output GV window is selected, and then select Results →

Summary. The final electronic energy should be reported along with other useful

information such as the point group. Fill in the following two tables.

Gaussian Calculation Tables

Energy of Optimized Monomers (in hartrees):

Water ( H2O )

Methanol ( CH3OH )

Dimethyl Ether ( (CH3)2O )

Energies of Optimized Complexes (in hartrees):

Water Methanol Dimethyl ether

Water

Methanol

Dimethyl Ether

Interaction Energies:

( ) ( ) ( )E E AB E A E BΔ = − −

Remember to subtract 2 individual molecules from the complex (it can be 2 of the

same molecule). Report your final numbers in hartrees and kcal/mol (1 kcal/mol =

627.5095 hartrees).

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Water Methanol Dimethyl ether

Water

Methanol

Dimethyl Ether

If you are diligent try this again changing the method (B3LYP, MP2, etc.) and/or

basis set (6-31G, 6-311G(d), aug-cc-pvdz, ect.). Just remember that the higher the level

of theory, the longer it takes for geometries to optimize.

These binding energies will give you the relative strengths of the hydrogen bonds

that occur within these molecules in the gas phase. Does the type of substituent play a

part in the strength of the hydrogen bond that is formed between the molecules? How

might this be explained? Think of the electronic nature of the substituent.

Conformational Isomerism in n-Butane

Here we will investigate the conformational isomers of n-butane by using

theoretical methods. To start this, we will build n-butane in GaussView and save the file

(Remember where you save it). We then will modify the z-matrix in the text input file,

in order to compute the potential energy surface associated with the C-C-C-C torsion

angle in n-butane.

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H

HH

H

H

H

H

C

H

C

H

C

C

H

C

CH3

H

HH

CH3

H

Shown here is the anti- or trans conformation, with the C-C-C-C dihedral angle at

180°. This is only one of the naturally occurring stable structures, the other being the

gauche conformation, with a dihedral of ~ 60. 0°. We will be able to see these

conformations as we plot the dihedral angle (in degrees) vs. the relative energy.

To get started we will need to modify the z-matrix within the text file in order to

compute the electronic energy at each corresponding dihedral angle. This will be done by

identifying the dihedral of interest within the z-matrix. Open the n-butane z-matrix file

with Notepad. First, HF/3-21G(d) level of theory will be replaced with B3LYP/ 6-

31g(d). Second, the input file will need to eliminate the geometry connectivity. This is

done by eliminating the geom = connectivity command in the input deck and everything

after the last dihedral angle listed at the bottom of the z-matrix file. Geom=connectivity

must be replaced with opt=(z-matrix), to ensure the structure is optimized. Third, we

must identify the appropriate dihedral angle in which the corresponding rotational barrier

will be computed, and then alter the input accordingly. In the coordination section of the

z-matrix we see that the last carbon (C11) is bonded to atom 8 (C8), forming an angle

with atom 5 (C5), and forming a dihedral angle or torsion angle with atom 1 (C1),

represented by B10, A9, and D8, respectively. This is the dihedral angle we are interested

in (C11-C8-C5-C1). Within the z-matrix you should see variables listed after the

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coordination input (B1, B2, … A1, A2, ... D1, D2, ...etc.). The letters B, A, and D,

represent bonds, angles, and dihedrals, respectively. Close to the end of the variable list

you should see D8 ≈ 180.0o (the variable representing the dihedral angle). Replace “D8 ≈

180.0” with “D8 0.0 s 6 30.0”. This line will set D8 equal to 180.0 and scan (s for

scan) the dihedral angle by 30.0o a total of six times. Consequently the structure will be

optimized with the dihedral angle fixed at 0.0, 30.0, 60.0 ….etc.

Now we are going to compute the potential energy surface associated with the C-

C-C-C dihedral angle. Open up the Guassian 3 which opens up a window. Go to File →

Open and open the n-butane file, which displays the contents of that file, press run which

is at the right hand corner of the window.

When the simulation is complete, select File → Open in the Guassview main

window. Select the output file to open and check the Read Intermediate Geometries box

at the bottom of the screen. You should see a scroll at the top left corner of the structure

window, allowing you to scroll through the seven output structures (one for each dihedral

angle analyzed). To determine the electronic energy for each structure, follow the

instructions given in the previous section. When the summary box is displayed you can

view the electronic energy for each structure by scrolling through the different structures

in the GV window. Convert the electronic energies that are in hartrees to kcal/mol and

subtract off the lowest energy. You can plot the relative energy vs. torsion angle in Excel

or another similar program. Also, you may visualize the potential energy surface by

clicking Results → Scan.

Fill in the following plot and turn it in with the tables filled out within this tutorial.

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How do the different conformations affect the relative energy? Does the plot

strengthen or weaken arguments on how steric effects affect the conformational

configuration that we learn about in Organic Chemistry class?

Glycine in the Gas Phase and in Water

Glycine is the most simple amino acid, yet it has different structural forms

depending on external environmental influences, i.e. whether it is in the gas phase or in a

solution. In the gas phase, it has no charge separation and exists as Figure A below.

However, in water, it forms a zwitterion, with a deprotonation at the oxygen followed by

a protonation at the nitrogen, as shown in Figure B.

0 30 60 90 120 150 180

C-C-C-C dihedral angle (o)

Rel

ativ

e E

nerg

y (k

cal/m

ol)

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H

N

CC

HH

O

H

OH

H

N

CC

HH

O

OH

H +

-

Figure A Figure B

In this section, we will investigate the effects of solvation on glycine and look to

see how the charge is distributed throughout the molecule. First start by building glycine

and it’s zwitterions in separate GV windows. We will optimize these at the HF/3-21+G*

level of theory with no solvation. Then optimize them again with a method of solvation

and the same level of theory, creating a four separate outputs. For the solvation method,

we will use the IEFPCM method. Fill in the Table below to see how the energies and

charge distributions are affected by the solvent.

Energy

(hartress)

Energy

(kcal/mol)

Dipole

moment

CO bond

lengths

NH bond

lengths

Carbonyl C

charge

Nitrogen

charge

Glycine

Glycine

zwitterion

Glycine

w/

solvation

Glycine

zwitterion

w/

solvation

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What are the energies associated with solvating the glycine and the glycine

zwitterions? (Hint: Just subtract the energy of the molecule from the energy of the

solvated molecule.) Does the result support our understanding of glycine and its

zwitterion’s capabilities of being solvated, i.e. is it easier to solvate the zwitterion?

To see how the charge is distributed in the zwitterions, we will look at the

individual isolated ions found within. Build separate molecules in GaussView for the

methylammonium cation and the acetate anion. Examples are shown below in Figures C

and D.

CC

HH

O

O

H

H

N

CH

H

H

H

H-

+

Figure C Figure D

We will optimize these at the same level of theory and look at the charges on the

protonation and deprotonation sites. We will also make a comparison of the NH and CO

bond lengths, to the zwitterions. Fill in the chart below to make the comparison.

CO bond

lengths

NH bond

lengths

Carbonyl

C charge

Oxygen

charge

Nitrogen

charge

Glycine

Glycine zwitterion

Methylammonium cation x

Acetate anion x

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Are the structures of the individual ions closer to that of the glycine or the glycine

zwitterion? Is their a full charge separation in the zwitterion based on this comparison?

How might we improve on these results?

II.) Molecular Dynamic Calculations

** Turn in the answers to the questions on a separate piece of paper.

Ramachandran Plot and Molecular Dynamics of Alanine Dipeptide (MOE)

In this Molecular Dynamics (MD) section, we will look at how a molecule

explores the energy landscape in both the gas phase and in solution. We will notice what

areas of the energy surface will be changed the most and how this affects the path of the

dipeptide in the course of the simulation.

For the MD study of conformations, we will do simulations in solution and in the

gas phase. For the MD simulations, we will look at the blocked alanine dipeptide.

1.) The contour map (Ramachandran plot) of the dihedral angles in the peptide. This will

show us which conformation(s) the dipeptide will mostly like be situated in (with respect

to the (phi/psi ) φ/ψ angles).

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Blocked alanine dipeptide can be built in MOE by using the Edit→Build → Protein tab.

Then just click on alanine (ALA) and select both the Acetylate N-Term and Amidate C-

Term tabs at the top of the screen.

Make the φ/ψ (Ramachandran) plot for the blocked alanine dipeptide. Use the

Compute→Mechanics→Dihedral Contour Plot option. Select the φ angle (CO-N-Cα-CO

) first by clicking on the four atoms and then the ψ angle (N-Cα-CO-N) the same way. Do

this for the different force fields (Edit → Potential setup → Load ).

Go to the Pitt library website and get the following reference:

Head-Gordon, T., et. al. J. Am. Chem. Soc. 1991, 113, 5989-5997.

Compare your φ/ψ Plot to the plot found in the paper. See which force field gives the best

results. You may want to try other amino acids and compare the φ/ψ maps. What features

are the same; what features are different?

2.) To do the molecular dynamics simulation, go to Compute→ Simulations→

Dynamics (be sure to click the box to open the Database Viewer). Do the simulations

for the gas phase (with no solvation) and in solution for 100 picoseconds apiece. Set the

number of picoseconds to 100 by typing the corresponding number in the Run box. Do

the solution simulations twice, one with explicit (build a water box around dipeptide).

Draw the dipeptide for each simulation afresh as mentioned above and then simulation.

Simulation with explicit waters

To add explicit waters you have to generate water box by clicking Edit → Build→ Water

Soak. Several options will be available for you to explore. Toggle on update Periodic

Box and for this case click on Box and then adjust Box size. This will create a box

around your system of the appropriate size automatically! Then Compute the dynamics

simulation by following the steps mentioned in the previous simulation with a different

name.

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Simulation with implicit waters

To add implicit water go to Edit →Potential setup. Under the solvation term the

Born model should be applied and. Fix hydrogens and charges and run the simulation

again.

In the main screen click Close on the right side and discard the data. In the

Database viewer, go to File → Browser (the structure should reappear in the main

screen). In the main screen, click on Measure → Dihedrals to measure the dihedrals

(both the φ and ψ angles). As you move through the simulations by advancing the frames

one or more than one at a time (you will see how in the Dynamics Animator), follow the

φ/ψ angles in the in Dihedral contour plot that was made in the beginning of this exercise.

Click on Compute → Conformations → Geometry in the database viewer window. Now

hold the shift button and select four atoms (N-C-Cα-N) in viewer. The Dihedral field in

the Conformation geometries opened panel will be shown with the selected atoms and

click Measure. Dihedral angles for the different conformers in the data base are

calculated. Click on Display → Plot and a panel opens up in the bottom half of the

database. Go to that particular column under Plot Fields. Notice how the different

conformers are plotted according to the dihedral angles calculated. Drag the mouse over

the plot for a particular conformer. This selects the conformer in the upper part of

database so that we can look at it specifically.

Does the contour map change from going from the gas phase to solution phase

(yes, you need to make another contour plot for the solution phase blocked alanine

dipeptide)? What factors do you think influences the change(s) in the energy landscape

and the path of the dipeptide through the simulation?

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Docking Simulations (MOE)

In the Docking exercise, we will show you how to dock a drug/inhibitor (or

anything else) into the active site or the proposed (binding site) active site of a molecule.

From the earlier labs, we will show the factors that play a part in the stability of the

docked structure and how you might be able to enhance the structure to find more

favorable interactions. An example of this might be modifying a drug so it binds better in

an active site.

For the docking example, we will use carbonic anhydrase II with an inhibitor

(your choice of inhibitor when downloading (examples: PDB Id’s for a sulfonamide is

1BCD and aromatic is 1CRA ))

Open the downloaded PDB. A window will appear with some options. Leave all

the defaults and continue. Now let us prepare the protein by adding hydrogens, removing

water molecules. This can be easily done in the Sequence Editor (Click SEQ in the

upper right hand corner). Also, make the drug a separate chain by opening the Sequence

Editor selecting Edit → Split. Remove water molecules by Right Clicking on the water

chain in the Sequence Editor and selecting delete. Add hydrogens and charges by having

the substrate selected and going to Edit→Potential Setup, click on Fix hydrogens and Fix

charges. Select a force field in the Potential Control under Load. In this case, use the

MMFF94 force field. Go to Compute→Simulations→Dock. Provide the name of db to

be generated. Select receptor atoms under receptor, select Ligand atoms under Site

and Ligand atoms under Ligand. Leave default Alpha triangle under Placement as

such and choose Affinity dG under Rescoring 1. Leave the remaining two choices of

Refinement and Rescoring 2 to None and click OK at the bottom of screen.

Analyze the results by looking at the S, energy values, pocket surface, H bonds,

etc. Clicking Display → Plot allows certain columns to be plotted. In addition you may

click File → Browser and scroll through the different properties or structures (click on

the Subject box to see the properties). Open the sequence editor by clicking SEQ at the

top right hand corner. Go to selection and click on Synchronize and then select the ligand

in SEQ. This selects the ligand in the viewer (black window). Click Compute →

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Surfaces and Maps to display the binding pocket. Select the option Selected Atoms

under Near in Surfaces and Maps and click Apply. Right now you should be thinking

“Oooooo Pretty Colors.” Modify the substrate by utilizing the Builder tool on the right

side of the screen and repeat the docking process to determine whether the new substrate

binds better or worse.

MOE allows one to see an electrostatic contour of a section so we may make an

educated guess on a modified substrate. To generate those maps, go to Compute →

Surfaces and Maps and select Electrostatic map under Surface and Selected Atoms

under Near and click apply. It generates maps in the viewer. White regions are electro

statically neutral regions whereas negative regions are shown red (acceptor) and positive

regions are shown blue (donor). Use your chemical intuition to make choices about how

to modify the substrate. Many websites offer information on the hydrophobic/

hydrophilic nature of amino acids, if that is not your area of expertise).

Write down the structure of the new compound that you have made. Does this

new molecule seem to work better or worse at binding to the Carbonic Anhydrase II?

What would be other considerations for making a “new” drug to bind in the active site

and prevent the enzyme from working properly?

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III.) Molecular Dynamic Simulations (NAMD)

In this exercise you will simulate the protein’s motion in vaccum as well as

solvent/water by using a molecular dynamics program called NAMD. We will compare

and contrast the two simulations. A CD will be provided to you with the necessary files.

Simulation of Bovine Pancreatic Trypsin Inhibitor (bpti) in vacuum.

Use the bpti.pdb provided to you in visualization tutorial. The next step is to

create a principle structure file (psf) using the psfgen program. Make sure bpti.pdb,

bpti.pgn.txt, top_all27_prot_lipid.inp are in the same folder. Load the bpti protein in

VMD by Clicking File → New Molecule → Browse. Next, go to the directory where

the files mentioned previously are stored in the VMD command window/(black). Once

you are in that directory, type the following line in that window and press enter.

psfgen bpti.pgn.txt

If everything is done correctly, the bpti.pdb will be modified and a bpti.psf will be

generated.

Open up the command prompt (CMD) by typing cmd in run window (Start → RunI).

Go to your working directory (the directory where your files are)and type the following.

While typing the following keep in mind that [] means a single space.

namd2[]vacuum.conf []>[]bpti_vac.log . (Use this if your pc has one Processor)

namd2 []+p2[]vacuum.conf []>bpti_vac.log (Use this if your pc has two Processors)

This will take about ten minutes. If successful a number of different files named

beginning with bpti and different extensions will be created.

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Load the bpti.pdb in VMD. Left Click on loaded bpti.pdb in the VMD main window to

highlight it and then Right click to select Load Data into molecule. Load bpti.dcd file, a

trajectory file. Click on play and see how the protein moves throughout the simulation.

Open up NAMD plot under Extensions → Analysis → NAMD plot. Load the

bpti_vac.log file created in NAMD plot and select the Energies → Total, kinetic etc.

Simulation of Bovine Pancreatic Trypsin Inhibitor (bpti) in water

Close VMD and start again or delete the previous files and do the following.

Use the psf file generated in the previous simulation. For solvating the protein

Copy the psf file generated in previous simulation to the water folder where you have the

pdb file. Load the pdb file into VMD and right click on loaded bpti.pdb and select Load

Data into molecule to load bpti.psf file. Go to your directory in the VMD command

window and type the following:

package[]require[]solvate

Solvate[]bpti.psf[]bpti.pdb[]–t[]5[]–o[]bpti_wb

This creates 5 Å3 water box around the protein and two new files are generated:

bpti_wb.psf and bpti_wb.pdb. These files will be automatically loaded into VMD and

you should see a water box surrounding the protein. If you don’t see the water box load,

delete the previously loaded bpti.pdb and bpti.psf files and load the generated

bpti_wb.pdb and right click on loaded bpti_wb.pdb and select load data into molecule

and load bpti_wb.psf file.

To simulate we now need the box dimensions in the bpti_water.config file. To

find those dimensions type the following commands in the VMD tk window which can

be found under Extensions→ Tk Console.

set everyone [atomselect top all] selects all the atoms.

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measure minmax $everyone reports the dimensions of the box.

Subtract the minimum value from the maximum value, the result will be your box

dimensions.

measure center $everyone provides the coordinates of the center of the box.

You will be copying these dimensions into the periodic boundary conditions section of

the bpti_water_npt.conf file. The x value goes in the top row and the rest of the numbers

in that row will be zero. In the middle row everything thing will be zero except for the

middle number and all zeroes except the last number in the last row. Now copy the center

values calculated under the cell origin and save the file.

Open up the command prompt (CMD) and go to your working directory type the

following and enter

namd2[]bpti_water_npt.conf []>[]bpti_water.log . (Use this if your pc has one

Processor)

namd2 []+p2[]bpti_water_npt.conf []>[]bpti_water.log (Use this if your pc has two

Processors)

Running NAMD creates number of different files named as bpti with different extensions

Load the bpti_wb.pdb in VMD. Right click on loaded bpti_wb.pdb and select load data

into molecule and select bpti_wb_eq.dcd file, a trajectory file. Click on play and see

how the protein moves throughout the simulation.

Open up NAMD plot under Extensions → Analysis → NAMD plot. Load the created

bpti_water_npt in NAMD plot and select the energies → total, kinetic etc. The plots

can be exported to postscript viewer programs to use later.

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