protein sturcture prediction and molecular modelling
TRANSCRIPT
PRESENTED BY:P. DILEEPB.Pharmacy
M.Pharmacy(2ND sem)SHIFT II, Roll no:30Department of PharmacologyVAAGDEVI COLLEGE OF PHARMACY
Contents of Seminar2
Introduction
Molecular modeling
Types of molecular modeling
Applications of molecular modeling
Proteins in brief
Purpose of protein structure prediction
Types of PSP
Conclusion
3
Molecular Modeling4
The science (or art) of representing molecular structures numerically and simulating their behavior with the equations of quantum and classical physics.
Combination of computational chemistry and computer graphics. Allows scientists to generate and present molecular data including
geometries (bond lengths, bond angles, torsion angles), energies (heat of formation, activation energy, etc.), electronic properties (moments, charges, ionization potential, electron affinity), spectroscopic properties (vibrational modes, chemical shifts) and bulk properties (volumes, surface areas, diffusion, viscosity, etc.).
Thomas L L, David A W, Victoria K(1999), Foye’s principles of Medicinal Chemistry, Lippincott Williams & Wilkins publications, 6th edition, 3, pp. 55-63.
5
Potential energy variation
Thomas L L, David A W, Victoria K(1999), Foye’s principles of Medicinal Chemistry, Lippincott Williams & Wilkins publications, 6th edition, 3, pp. 55-63.
Molecular Modeling methods
6
The two most common computational methods Molecular mechanics Quantum mechanics
Both these methods produce equations for the total energy(E) of the structure.
MOLECULAR MECHANICS: Calculation of energy of atoms, force on atoms and
their resulting motion. Used to model the geometry of the molecule, motion of
molecule and to get the global minimum energy structure.
Thomas L L, David A W, Victoria K(1999), Foye’s principles of Medicinal Chemistry, Lippincott Williams & Wilkins publications, 6th edition, 3, pp. 55-63.
Molecular mechanics7
Consider a molecule as system of rigid balls connected via springs.
Depends strongly on concepts of bonding Follows the Newtonian laws Neglect the electronic degrees of freedom
Leach A R(2001), Molecular Modelling: Principles and Applications, Oxford press, 4th edition, 1, pp. 1-3.
8
Potential surface
Study of force field
Study of Electrostatics
Molecular dynamics
Conformational Analysis
Methods for Molecular mechanics study
Leach A R(2001), Molecular Modelling: Principles and Applications, Oxford press, 4th edition, 1, pp. 1-3.
Methods for Molecular mechanics study
9
Force field is used to describe the total potential energy of a molecule or system as a function of geometry. and the set of parameters required is called “force field parameters”. The total energy is a sum of Taylor series expansions for stretches for every pair of bonded atoms, and adds additional potential energy terms coming from bending, torsional energy, Vander wall energy,
electrostatics and cross terms.
Study of Electrostatics involves the study of interaction between various dipoles.
Leach A R(2001), Molecular Modelling: Principles and Applications, Oxford press, 4th edition, 1, pp. 1-3.
10
Leach A R(2001), Molecular Modelling: Principles and Applications, Oxford press, 4th edition, 1, pp. 1-3.
Methods for Molecular mechanics study
11
Molecular dynamics program allow the model to how the natural motion of atoms in the structure. This is achieved by including the kinetic energy term of atoms in the force field equation by using equations based on Newton's law of motion.
Conformational Analysis involves the determination or analysis of the spatial arrangement of the functional group of the respective molecule. Strategies used to study the conformational analysis are Rigid geometry approximation, Rigid body rotation, Conformational clustering.
Leach A R(2001), Molecular Modelling: Principles and Applications, Oxford press, 4th edition, 1, pp. 1-3.
QUANTUM MECHANICS12
Provides information about both nuclear position and distribution.
Based on study of arrangement and interaction of electrons and nuclei of a molecular system.
It does not require the use of parameters similar to those used in molecular mechanics.
It is based on the wave properties of electrons and all material particles.
Griffith S, David J(2004), Introduction to Quantum Mechanics, Prentice Hall press, 2nd edition, pp. 1-4.
13
QUANTUM MECHANICS
Where,H = Hamiltonian for the system,Ψ(“p-sigh”) = wave function,E = energy.Simply put, the Hamiltonian is an “operator,” a mathematical construct that operates on the molecular orbital, Ψ, to determine the energy.U= Potential energy,K= Kinetic energy.
H Ψ = E Ψ = (U+K) Ψ
Thomas L L, David A W, Victoria K(1999), Foye’s principles of Medicinal Chemistry, Lippincott Williams & Wilkins publications, 6th edition, 3, pp. 55-63.
ADVANTAGES14
To calculate the value of potentials, electron affinities ,heat of formation, dipole moment and other physical properties
To find the electron density in a structure
To determine the points at which a structure will react with electrophiles and nucleophiles
To determine the shape and electron density of a molecule
Rajkumar B, Branson K, Giddy J, Abramson D(2003), The Virtual Laboratory : A toolset to enable distributed molecular modeling for drug design on the World-Wide Grid, Concurrency and computation, 15, pp. 1–25.
15
Proteins….16
http://courses.washington.edu/conj/protein/insulin2.gif
http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html
If there is a job to be If there is a job to be done in the molecular done in the molecular world of our cells, world of our cells, usually that job is done usually that job is done by a protein.by a protein. CATALASE
An enzyme which removes Hydrogen peroxide from your body so it does not become toxic
A protein hormone which helps to regulate your blood sugar levels
Proteins for cell motility 17
Myosin and actin filaments work in coordination for the proper muscle contraction
http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html
18
Eukaryotic exoskeleton
Tubulin frame work for the exoskeleton
Microtubules
Cellular coat
http://www.fz-juelich.de/ibi/ibi-1/Cellular_signaling/http://cpmcnet.columbia.edu/dept/gsas/anatomy/Faculty/Gundersen/main.html
Cell structures
http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html
19
Energy
Progress of reaction
Substrate Product
+2 2
http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html
Enzymes
20
http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html
Harmones and channels
21
http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html
Immune response
Proteins can be fibrous or Proteins can be fibrous or globularglobular
22
Fibrous proteins23
http://opbs.okstate.edu/~petracek/2002%20protein%20structure%20function/CH06.gif
http://my.webmd.com/hw/health_guide_atoz/zm2662.asp?printing=true
•Collagen is the most abundant protein in vertebrates. Collagen fibers are a major portion of tendons, bone and skin. Alpha helices of collagen make up a triple helix structure giving it tough and flexible properties.•Fibroin fibers make the silk spun by spiders and silk worms stronger weight for weight than steel! The soft and flexible properties come from the beta structure.•Keratin is a tough insoluble protein that makes up the quills of echidna, your hair and nails and the rattle of a rattle snake. The structure comes from alpha helices that are cross-linked by disulfide bonds.
Globular proteins24
Cell motility – proteins link together to form filaments which make movement possible.
Organic catalysts in biochemical reactions – enzymes
Regulatory proteins – hormones, transcription factors
Membrane proteins – protein channels
Defense against pathogens – poisons/toxins, antibodies, complement
Transport and storage – hemoglobin
http://my.webmd.com/hw/health_guide_atoz/zm2662.asp?printing=true
Molecular Logic of Life is SameMolecular Logic of Life is Same
Biochemically, all things living – animals, plants, bacteria, viruses, etc. – are remarkably similar
English
26-Letter alphabet Only one grammar Extremely diverse literature
Genome
4-Letter alphabet Only one grammar Extremely diverse organisms
25
G T A C T A
Chromosome
The order of bases in DNA is a code for making proteins. The code is read in groups of three
DNA
Gene
Cell machinery copies the code making an mRNA molecule. This moves into the cytoplasm.
Ribosomes read the code and accurately join Amino acids together to make a protein
AUGAGUAAAGGAGAAGAACUUUUCACUGGAUAM S E E LK G TF G
The protein folds to form its working shape
M
S EK G
E L TF G
M
S
E
K
GE L TF G
M
S
E
K
G
EL
TF
G
M
S
E
K
G
EL
TF
G
M
S
E
K
G
EL
T F
G
CELL
NUCLEUS
Gene Expression
26
M
S
E
K
G
EL
T F
G
T
GM
S
E
K
G
EL
F
T
G
M
E
K
G
EL
FS
http://my.webmd.com/hw/health_guide_atoz/zm2662.asp?printing=true
Hallmark of Proteins: SpecificityHallmark of Proteins: Specificity
Know exactly which small molecule (ligand) they should bind to or interact with.
Also know which part of a macromolecule they should bind to.
27
Function is critically dependent on Function is critically dependent on structurestructure
One Aspect of Genome Sequence Analysis is to One Aspect of Genome Sequence Analysis is to Assign Functions to ProteinsAssign Functions to Proteins((Reverse GeneticsReverse Genetics))
Schween G, Egener T, Fritzkowsky D, Granado J, Guitton M C(2005), Large-scale analysis of Physcomitrella plants transformed with different gene disruption libraries: Production parameters and mutant phenotypes, Plant Biology, 7 (3), pp. 228–237.
28
Sequence
Structure Function
How Does Sequence Specify Structure?How Does Sequence Specify Structure?
Structure has to be determined experimentally
The Protein Folding ProblemThe Protein Folding Problem(second half of the genetic code)(second half of the genetic code)
??Functional Genomics
29
Protein Structure30
• Levels of organization– Primary Sequence– Secondary Structure (Modular building
blocks)• α-helices• β-sheets
– Tertiary Structure– Quartenary Structure
• Hydrophobic/Hydrophilic Organization.Lubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company
press, 5th edition, pp. 198-230.
31
Lubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company press, 5th edition, pp. 198-230.
Protein Structure
Secondary StructureSecondary Structure: -helix32
Lubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company press, 5th edition, pp. 198-230.
Secondary StructureSecondary Structure: -sheets33
Lubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company press, 5th edition, pp. 198-230.
Definition of -turn
34
four consecutive residues i, i+1, i+2 and i+3 that do not form a helix and the turn lead to reversal in the protein chain.
The conformation of -turn is defined in terms of two central residues, i+1 and i+2 and can be classified into different types on the basis of this conformation.
i
i+1 i+2
i+3H-bond
D <7ÅLubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company press, 5 th edition,
pp. 198-230.
Biology/Chemistry of Protein Structure
35
Primary
Secondary
Tertiary
Quaternary
Assembly
Folding
Packing
Interaction
S T
R U
C T
U R
E P R
O C
E S
S
Lubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company press, 5 th edition, pp. 198-230.
3 main questions…3 main questions…36
1. Why predict the structure?
2. Methods for structure prediction
3. What next?
Purpose of PSP
Explaining phenotype of existing mutations (experimental or patient-derived)
Designing mutants to disrupt or alter specific functions (leaving others unaffected)
Hints at function
Drug design (at high sequence identity)
Hypothesis generation
37
Mateusz K, Michał J, Andrzej K(2011), Multiscale Approaches to Protein Modeling, Springer Science Business Media, 12th edition, pp. 21-32
• Anfinsen’s (1973) thermodynamic hypothesis:
Proteins are not assembled into their native structures by a biological process, but folding is a purely physical process that depends only on the specific amino acid sequence of the protein.
38
Anfinsen C B(1973), Principles that govern the folding of protein chains, Biological Science, 181, pp. 223–230
The Prediction Problem39
Can we predict the final 3D protein structure knowing only its amino acid sequence?
• Studied for 4 Decades
• “Holy Grail” in Biological Sciences
• Primary Motivation for Bioinformatics
• Based on this 1-to-1 Mapping of Sequence to Structure
• Still very much an OPEN PROBLEMMateusz K, Michał J, Andrzej K(2011), Multiscale Approaches to Protein Modeling, Springer Science Business Media, 12th edition, pp. 21-32
PSP: Major Hurdles40
Energetics We don’t know all the forces involved in detail Too computationally expensive BY FAR!
Conformational search impossibly large 100 AA. protein, 2 moving dihedrals, 2 possible positions
for each diheral: 2200 conformations! Levinthal’s Paradox
Longer than time of universe to search Proteins fold in a couple of seconds??
Mateusz K, Michał J, Andrzej K(2011), Multiscale Approaches to Protein Modeling, Springer Science Business Media, 12th edition, pp. 21-32
PSP: Goals41
Accurate 3D structures. But not there yet. Good “guesses”
Working models for researchers Understand the FOLDING PROCESS
Get into the Black Box Only hope for some proteins
25% won’t crystallize, too big for NMR Best hope for novel protein engineering
Drug design, etc.
Mateusz K, Michał J, Andrzej K(2011), Multiscale Approaches to Protein Modeling, Springer Science Business Media, 12th edition, pp. 21-32
Comparative Modeling--Basic Protocol
1.Identification of homologue for target sequence2.Alignment of target sequence to template sequence and structure3.Side-chain modeling, copy the backbone of the template and model the new side chains onto this backbone4.Loop modeling, for insertions and deletions in the alignment5.Refinement of model -- moving template closer to target6.Assessment of (predicted) model quality7.Using the model to explain experiments and guide new ones
42
David F B, Charlotte M D, Hampapathalu A N, Nuria C, An Iterative Structure-Assisted Approach to Sequence Alignment and Comparative Modeling, PROTEINS: Structure, Function, and Genetics Supplementations, 3, pp. 55-60.
43
Experimental techniques for structure Experimental techniques for structure determinationdetermination
X-ray Crystallography Nuclear Magnetic Resonance
spectroscopy (NMR)
Electron Microscopy/Diffraction
Free electron lasers.
David F B, Charlotte M D, Hampapathalu A N, Nuria C, An Iterative Structure-Assisted Approach to Sequence Alignment and Comparative Modeling, PROTEINS: Structure, Function, and Genetics Supplementations, 3, pp. 55-60.
44
From small molecules to viruses Information about the positions of
individual atoms Limited information about dynamics Requires crystals
X-ray CrystallographyX-ray Crystallography
Saville W B, Shearer G (1925), An X-ray Investigation of Saturated Aliphatic Ketones, Journal of the Chemical Society, 127, pp. 591.
45
NMRNMR
Limited to molecules up to ~50kDa (good quality up to 30 kDa)
Distances between pairs of hydrogen atoms
Lots of information about dynamics Requires soluble, non-aggregating
material Assignment problem
Addess M, Kenneth J, Feigon J(1996). Introduction to 1H NMR Spectroscopy of DNA. Bioorganic Chemistry: Nucleic Acids, Oxford University Press, 8th edition, pp. 238.
46
Electron Microscopy/ DiffractionElectron Microscopy/ Diffraction
Low to medium resolution Limited information about
dynamics Can use very small
crystals (nm range) Can be used for very large
molecules and complexes
Tertiary Structure Prediction47
Template Modeling Homology Modeling Threading
Template-Free Modeling ab initio Methods
Physics-Based Knowledge-Based
Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288.
HOMOLOGY MODELING
48
Constructing an atomic-resolution model of the "target" protein from its amino acid sequence and an experimental 3d structure of a related homologous protein (the "template").
Homology modeling relies on the identification of one or more known protein structures likely to resemble the structure of the query sequence, and on the production of an alignment that maps residues in the query sequence to residues in the template sequence
This approach can be complicated by the presence of alignment gaps (commonly called indels) that arise from poor resolution in the experimental procedure (usually X-ray crystallography).
Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288
49
Homology modeling can produce high-quality structural models when the target and template are closely related, which has inspired the formation of a structural genomics consortium.
The analysis and prediction of loop structures for small and medium sized loops and the positioning of side chains, given the conformation of the protein's backbone.
HOMOLOGY MODELING
Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288
Threading or Fold Recognition Method
50
Computational protein structure prediction Distinction between two fold recognition scenarios. “Threading" (i.e. placing, aligning) each amino acid in the target
sequence to a position in the template structure, and evaluating how well the target fits the template. After the best-fit template is selected, the structural model of the sequence is built based on the alignment with the chosen template.
Homologous folds share the Same structure through divergent evolution from a common ancestor.
Analogous folds, on the other hand, share the same structure, but give insufficient evidence for an evolutionary relationship.
Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288
51
One popular model for protein folding assumes a sequence of events:
Hydrophobic collapse
Local interactions stabilize secondary structures
Secondary structures interact to form motifs
Motifs aggregate to form tertiary structure
Threading or Fold Recognition Method
Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288
52
It calculates energetics involved in the process of folding
Finding the structure with lowest free energy It is based on the ‘thermodynamic hypothesis’, which states that the
native structure of a protein is the one for which the free energy achieves the global minimum.
2 components to ab initio prediction:1. devising a scoring (ie, energy) function that can
distinguish between correct (native or native-like) structures from incorrect ones.
2. a search method to explore the conformational space. The most difficult, but most useful approach.
Ab-initio method
Richard B, David B(2001), AB INITIO PROTEIN STRUCTURE PREDICTION: Progress and Prospects, Annual review of biophysical and biomolecular structures, 30, pp. 73-88.
53
Ab-initio method
Sequence
Secondary structure
Prediction
Tertiary structure
Low energy structures
Predicted structureEnergy
Minimization
Validation
Mean field potentials
Richard B, David B(2001), AB INITIO PROTEIN STRUCTURE PREDICTION: Progress and Prospects, Annual review of biophysical and biomolecular structures, 30, pp. 73-88.
• Existing SSP Methods• Statistical Methods (Chou,GOR)• Physio-chemical Methods• A.I. (Neural Network Approach)• Consensus and Multiple Alignment
• Our Method APSSP of SSP• Neural Network• Example Based Learnning• Multiple Alignment
• Steps involved in APSSP• Blast search against protein sequence (NR)• Multiple Alignment (ClustalW)• Profile by HMMER, Result by Email
• Recogntion: CASP,CAFASP,LiveBench, MetaServer
Secondary Structure Prediction54
Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288
Web servers for structure prediction
JPRED:-http://www.compbio.dundee.ac.uk/~www-jpred/
PHD:-http://cubic.bioc.columbia.edu/predictprotein/
PSIPRED:-http://bioinf.cs.ucl.ac.uk/psipred/
Chou and Fassman:-http://fasta.bioch.virginia.edu/fasta_www/chofas.htm
55
Future technologies
Modeling of biologically relevant states of proteins using all available templatesHomooligomers
Heterooligomers
Amino acid modifications
Bound ligands (small molecules, nucleic acids)
Modeling of specific classes of proteins
Antibodies
Repeat proteins (ARM/HEAT, WD repeats)
56
Ram S, Yu Xia, Enoch H, Michael L(1999), Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, PROTEINS: Structure, Function, and Genetics, 3, pp. 194–198.
Available databases, software, and services
Rotamer library
ProtBuD -- biological units database across families
PISCES -- non-redundant sequences in PDB
MolIDE 1.5
ArboDraw -- drawing phylogenetic trees
BioDownloader -- automatic updating of biological databases
57
Ram S, Yu Xia, Enoch H, Michael L(1999), Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, PROTEINS: Structure, Function, and Genetics, 3, pp. 194–198.
Assessment of accuracy of PSP
P = (N – total incorrect) N
total incorrect = total number of residues whose conformations are predicted incorrectlyN = the number of residues in the protein.
58
Ram S, Yu Xia, Enoch H, Michael L(1999), Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, PROTEINS: Structure, Function, and Genetics, 3, pp. 194–198.
Applications of PSP:
Drug targetting. Pharmacogenetics. Pharmacogenomics. MOA. Dosage regimen.
59
Conclusion
Pharmacist
60
Biotechnologist
Molecular modeling
61
Conclusion
Pharmacist Biotechnologist
Protein structure prediction
References:References:
1. Lubert S, Tymoczko J L, Jeremy M B(2003). Text book of biochemistry, W. H. Freeman and company press, 5th edition, pp. 198-230.
2. Thomas L L, David A W, Victoria K(1999), Foye’s principles of Medicinal Chemistry, Lippincott Williams & Wilkins publications, 6th edition, 3, pp. 55-63.
3. Mateusz K, Michał J, Andrzej K(2011), Multiscale Approaches to Protein Modeling, Springer Science+Business Media, 12th edition, pp. 21-32.
4. Zhan Y Z, Tom L B(1996), The Use of Amino Acid Patterns of Classified Helices and Strands in Secondary Structure Prediction, Journal of Molecular biology, 260, pp. 61–76.
5. Schween G, Egener T, Fritzkowsky D, Granado J, Guitton M C(2005), Large-scale analysis of Physcomitrella plants transformed with different gene disruption libraries: Production parameters and mutant phenotypes, Plant Biology, 7 (3), pp. 228–237.
6. David F B, Charlotte M D, Hampapathalu A N, Nuria C, An Iterative Structure-Assisted Approach to Sequence Alignment and Comparative Modeling, PROTEINS: Structure, Function, and Genetics Supplementations, 3, pp. 55-60.
7. Thomas L, Ralf Z(2000), Protein structure prediction methods for drug design, Briefings in Bioinformatics, 3, pp. 275-288.
8. Anfinsen C B(1973), Principles that govern the folding of protein chains, Biological Science, 181, pp. 223–230.
62
References:References:
9. Richard B, David B(2001), AB INITIO PROTEIN STRUCTURE PREDICTION: Progress and Prospects, Annual review of biophysical and biomolecular structures, 30, pp. 73-88.
10. Saville W B, Shearer G (1925), An X-ray Investigation of Saturated Aliphatic Ketones, Journal of the Chemical Society, 127, pp. 591.
11. Addess M, Kenneth J, Feigon J(1996). Introduction to 1H NMR Spectroscopy of DNA. Bioorganic Chemistry: Nucleic Acids, Oxford University Press, 8 th edition, pp. 238.
12. Ram S, Yu Xia, Enoch H, Michael L(1999), Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, PROTEINS: Structure, Function, and Genetics, 3, pp. 194–198.
13. Rajkumar B, Branson K, Giddy J, Abramson D(2003), The Virtual Laboratory : A toolset to enable distributed molecular modeling for drug design on the World-Wide Grid, Concurrency and computation, 15, pp. 1–25.
14. Griffith S, David J(2004), Introduction to Quantum Mechanics, Prentice Hall press, 2nd edition, 1, pp. 1-4.
15. Leach A R(2001), Molecular Modelling: Principles and Applications, Oxford press, 4th edition, 1, pp. 1-3.
16. LEONOR C H, PAULO A S(2001), Protein folding : thermodynamic versus kinetic control, Journal of biological physics, 27, pp. 6-8.
63
64
Useful links:Useful links:
Date: 28/10/2012.1. http://dunbrack.fccc.edu2. http://courses.washington.edu/conj/protein/insulin2.gif3. http://www.biochem.ucl.ac.uk/bsm/pdbsum/1gwf/main.html4. http://opbs.okstate.edu/~petracek/2002%20protein%20structure%20function/CH06.gif5. http://my.webmd.com/hw/health_guide_atoz/zm2662.asp?printing=true6. http://www.grin.com/object/
external_document.274822/5fbac5ddfea3cb2dd3dde8ad8ee981f9_LARGE.png
65
Thank you…..Thank you…..