Classification of Protein Complexes
based on Biophysics of Association
Sandor Vajda
Boston University
“Tell me with whom you go,
and I'll tell you what you are.” Italian Proverb
List of Interactions
“FYI” filtered yeast interactome (Vidal 2004):
• involves ~1500 proteins,
• making ~2500 physical interactions
Structure: Nature of
Intreractions
PDB:
~ 25’000 solved crystal structures;
~ 10% complexes
computational
prediction of
structure and
specificity of
protein – protein
complexes
H. Jeong et al, Nature 2001
“Tell me how you contact your partners,
and I'll tell you who you are.”
Protein-protein docking


How proteins interact with each other?
Docking problem
 Predict docking configuration from the structures of
component proteins
 Bound vs. unbound docking
 Conformational change
Bound vs.unbound:
at least side chain conformations change
Fine details
Receptor
Ligand
Coarse details
Trypsin/APPI
Talk outline
1. What is the current state of docking?
2. What docking calculations tell us about the
nature of protein - protein complexes?
3. How to deal with side chain flexibility?
Proteins: Basics
CASP
CAPRI
ADEFFGKLSTKK…….
Sequence
O
O
N
...
N
N
O
O
Monomers
N
...
O
O
N
Building Blocks:
backbone & side chains
de novo
Rigid body degrees of
freedom
3 translation
3 rotation
docking
Structure Prediction
Structure
Complex
+
Benchmark set of protein complexes:
Chen, R. et al. (2003) A protein-protein docking benchmark. Proteins,
52, 88-91.




22 enzyme-inhibitor
19 antigen-antibody
11 “other” types
7 “difficult” cases
Comeau, S. et al. (2003) ClusPro: An automated docking and discrimination
method for the prediction of protein complexes. Bioinformatics, 20, 45-50.
Chen, R. et al. (2003) ZDOCK: An initial-stage protein-docking algorithm
Proteins, 52, 80-87
Li, L. et al. (2003) RDOCK: Refinement of rigid-body protein docking
predictions. Proteins, 53, 693-707.
Gray, J.J. et al. (2003) Protein–protein docking with simultaneous optimization
of rigid-body displacement and side-chain conformations. J. Molec. Biol.
331, 281-299
How current protein docking programs work?
Rigid Body Search
Select docked structures
with low energy
Cluster retained
conformations
Refine structures
Flexible side
chains
Filter 1: 20,000
Filter 2: 2,000
Filter 3: 30
Filter 4: 1?
Submit 10
models to
CAPRI
Algorithms of the 3 docking methods
Method
Step 1: Rigid body search
(Investigator)
Step 2: Rescoring, ranking, filtering,
and refinement
ClusPro
(Camacho
and Vajda)
Fast Fourier Transform (FFT)
correlation approach using
ZDOCK or DOT
Re-scoring with empirical potentials
and clustering
Gray and
Baker
Monte-Carlo search using
simplified protein geometry
and scoring function
Iterative repacking of side chains
and rigid-body docking repeated
until convergence. Final selection
by clustering.
ZDOCK
(Weng)
FFT correlation with shape
complementarity,
electrostatics, and
desolvation
FFT correlation with shape
complementarity
Clustering of conformations to
avoid redundancies
RDOCK
(Weng)
Re-scoring with empirical potentials
Effect of the interface area
uncertain
easy
difficult
very difficult
GOOD
Effect of hydrophobicity
uncertain
easy
-4
Size vs. Hydrophobicity
Type IV
difficult
Type III
uncertain
Type II
easy
Type I
easy
Type V
difficult
Benchmark by type
Type
difficult
IV
difficult
Type V
difficult
Type III
uncertain
Type II
easy
Type I
easy
Desolvation free energy
Type IV
Difficult
Type III
Uncertain
Type II
Easy
Antibody/
Antigen
-4
Small
signalling
complexes
Type I
Easy
Large
multienzyme
complexes
Type II
Or
Type V?
Type V
Hopeless
Transitional
complexes with
substantial
conformational
change
Enzymes
1400
2000
3400
Interface Area
Table I. Major differences between enzyme-inhibitor and antibody-antigen complexes
Property
Enzyme-inhibitor complexes
Antibody-antigen complexes
Interface area DASA
1400 Å2 < DASA < 2000 Å2,
Possibly < 1400 Å2
Interface connectedness
Single patch
Frequently multiple patches
Interface shape
Convex-concave
Mostly planar
Binding free energy DG,
kcal/mol
-17.5 kcal/mol < DG < -13.0
kcal/mol
-13.0 kcal/mol < DG < -6.5
kcal/mol
% Nonpolar residues in
interface
61% nonpolar (can reach
71%)
51% nonpolar (can be as
low as 44%)
Desolvation free energy
Negative (favorable)
Positive (unfavorable)
Conformational change
Generally moderate
Can be substantial; loop
and/or hinge motion
Crystallographic water
positions
Around perimeter of
interface
Within the interface
Classification of complexes
Type
a
Conformational
change
Interface
DASAa
Hydrophobicity
I
Small (rigid
interface)
Standardb
II
Small
DASA >
2000 Å2
III
Moderate, but
larger than for
Type I
Standard
IV
Restricted to
side chains
DASA <1400 Weak; mostly
polar and
Å2
charge-charge
interactions
V
Substantial
backbone
change,
C RMSD > 2 Å
DASA >
2000 Å2
Docking
outcome
Successful,
unless key side
chains are in
wrong
conformations
Strong; the
convex-concave
interface
provides good
shape
complementarity
Unimportant
Successful
Variable, but
generally weak.
Charge-charge
interactions can
be strong
Generally
moderate
Unpredictable;
can be very
difficult, even
with know
hypervariable
regions of
antibody
Hits are found,
but are generally
lost in scoring
and ranking
Example
Trypsinogen and trypsin inhibitor (1cgi):
KD = 0.2 pM, DASA = 1950 Å2, and
DGdes = -18.3 kcal/mol. Most complexes of
enzymes with their protein inhibitors are
in this category
Ribonuclease a and ribonuclease
inhibitor (1dfj): DASA = 2580 Å2,
DGdes = 18.6 kcal/mol, DEelec=-63.9 kcal/mol
KD = 0.15 nM
Hyhel-5 Fab with lysozyme (1mlc):
KD = 126M, DASA = 1390 Å2, and DGdes = 3.84 kcal/mol. Most antibody – antigen
complexes are in this category
Ras and Ras interacting domain (1lfd)
KD = 2M, DASA = 1130 Å2, and DGdes = 3.6
kcal/mol. A number of weak complexes
are in this category
Rigid body
Cyclin A and cyclin-dependent kinase 2
methods seem
(1fin): KD = 47.6 nM, DASA = 3390 Å2, and
to always fail for DGdes = 4.7 kcal/mol
these complexes
ASA – Acessible Surface Area, bStandard interface: 1400 Å2 < DASA < 2000 Å2, c C RMSD -  carbon Root Mean Square Deviation
Type I:
Enzyme-Inhibitor Complexes
trypsin inhibitor variant 3
alpha-chymotrypsinogen
Interface in the complex of alpha-chymotrypsinogen with trypsin inhibitor
Table I. Major differences between enzyme-inhibitor and antibody-antigen complexes
Property
Enzyme-inhibitor complexes
Antibody-antigen complexes
Interface area DASA
1400 Å2 < DASA < 2000 Å2,
Possibly < 1400 Å2
Interface connectedness
Single patch
Frequently multiple patch
Interface shape
Convex-concave
Mostly planar
Binding free energy DG,
kcal/mol
-17.5 kcal/mol < DG < -13.0
kcal/mol
-13.0 kcal/mol < DG < -6.5
kcal/mol
% Nonpolar residues in
interface
61% nonpolar (can reach
71%)
51% nonpolar (can be as
low as 44%)
Desolvation free energy
Negative (favorable)
Positive (unfavorable)
Conformational change
Generally moderate
Can be substantial; loop
and/or hinge motion
Crystallographic water
positions
Around perimeter of
interface
Within the interface
Classification of complexes
Type
a
Conformational
change
Interface
DASAa
Hydrophobicity
I
Small (rigid
interface)
Standardb
II
Small
DASA >
2000 Å2
III
Moderate, but
larger than for
Type I
Standard
IV
Restricted to
side chains
DASA <1400 Weak; mostly
polar and
Å2
charge-charge
interactions
V
Substantial
backbone
change,
C RMSD > 2 Å
DASA >
2000 Å2
Docking
outcome
Successful,
unless key side
chains are in
wrong
conformations
Strong; the
convex-concave
interface
provides good
shape
complementarity
Unimportant
Successful
Variable, but
generally weak.
Charge-charge
interactions can
be strong
Generally
moderate
Unpredictable;
can be very
difficult, even
with know
hypervariable
regions of
antibody
Hits are found,
but are generally
lost in scoring
and ranking
Example
Trypsinogen and trypsin inhibitor (1cgi):
KD = 0.2 pM, DASA = 1950 Å2, and
DGdes = -18.3 kcal/mol. Most complexes of
enzymes with their protein inhibitors are
in this category
Ribonuclease a and ribonuclease
inhibitor (1dfj): DASA = 2580 Å2,
DGdes = 18.6 kcal/mol, DEelec=-63.9 kcal/mol
KD = 0.15 nM
Hyhel-5 Fab with lysozyme (1mlc):
KD = 126M, DASA = 1390 Å2, DGdes = -3.84
kcal/mol, DEelec = --21.4 kcal/mol, Most
antibody – antigen complexes are in this
category
Ras and Ras interacting domain (1lfd)
KD = 2M, DASA = 1130 Å2, and DGdes = 3.6
kcal/mol. A number of weak complexes
are in this category
Rigid body
Cyclin A and cyclin-dependent kinase 2
methods seem
(1fin): KD = 47.6 nM, DASA = 3390 Å2, and
to always fail for DGdes = 4.7 kcal/mol
these complexes
ASA – Acessible Surface Area, bStandard interface: 1400 Å2 < DASA < 2000 Å2, c C RMSD -  carbon Root Mean Square Deviation
Type III:
Antigen-Antibody Complexes
chicken lysozyme
Monoclonal antibody
fab d44.1
Interface in the complex of chicken lysozyme with antibody fab d44.1
Desolvation free energy
Type IV
Difficult
Type III
Uncertain
Type II
Easy
Antibody/
Antigen
-4
Small
signalling
complexes
Type I
Easy
Large
multienzyme
complexes
Type II
Or
Type V?
Type V
Hopeless
Transitional
complexes with
substantial
conformational
change
Enzymes
1400
2000
3400
Interface Area
Classification of complexes
Type
a
Conformational
change
Interface
DASAa
Hydrophobicity
I
Small (rigid
interface)
Standardb
II
Small
DASA >
2000 Å2
III
Moderate, but
larger than for
Type I
Standard
IV
Restricted to
side chains
DASA <1400 Weak; mostly
polar and
Å2
charge-charge
interactions
V
Substantial
backbone
change,
C RMSD > 2 Å
DASA >
2000 Å2
Docking
outcome
Successful,
unless key side
chains are in
wrong
conformations
Strong; the
convex-concave
interface
provides good
shape
complementarity
Unimportant
Successful
Variable, but
generally weak.
Charge-charge
interactions can
be strong
Generally
moderate
Unpredictable;
can be very
difficult, even
with know
hypervariable
regions of
antibody
Hits are found,
but are generally
lost in scoring
and ranking
Example
Trypsinogen and trypsin inhibitor (1cgi):
KD = 0.2 pM, DASA = 1950 Å2, and
DGdes = -18.3 kcal/mol. Most complexes of
enzymes with their protein inhibitors are
in this category
Ribonuclease a and ribonuclease
inhibitor (1dfj): DASA = 2580 Å2,
DGdes = 18.6 kcal/mol, DEelec=-63.9 kcal/mol
KD = 0.15 nM
Hyhel-5 Fab with lysozyme (1mlc):
KD = 126M, DASA = 1390 Å2, and DGdes = 3.84 kcal/mol. Most antibody – antigen
complexes are in this category
Ras and Ras interacting domain (1lfd)
KD = 2M, DASA = 1130 Å2, and DGdes = 3.6
kcal/mol. A number of weak complexes
are in this category
Rigid body
Cyclin A and cyclin-dependent kinase 2
methods seem
(1fin): KD = 47.6 nM, DASA = 3390 Å2, and
to always fail for DGdes = 4.7 kcal/mol
these complexes
ASA – Acessible Surface Area, bStandard interface: 1400 Å2 < DASA < 2000 Å2, c C RMSD -  carbon Root Mean Square Deviation
ribonuclease a
Ribonuclease inhibitor
Interface in the complex of ribonuclease a with ribonuclease inhibitor
Desolvation free energy
Type IV
Difficult
Type III
Uncertain
Type II
Easy
Antibody/
Antigen
-4
Small
signalling
complexes
Type I
Easy
Large
multienzyme
complexes
Type II
Or
Type V?
Type V
Hopeless
Transitional
complexes with
substantial
conformational
change
Enzymes
1400
2000
3400
Interface Area
Classification of complexes
Type
a
Conformational
change
Interface
DASAa
Hydrophobicity
I
Small (rigid
interface)
Standardb
II
Small
DASA >
2000 Å2
III
Moderate, but
larger than for
Type I
Standard
IV
Restricted to
side chains
DASA <1400 Weak; mostly
polar and
Å2
charge-charge
interactions
V
Substantial
backbone
change,
C RMSD > 2 Å
DASA >
2000 Å2
Docking
outcome
Successful,
unless key side
chains are in
wrong
conformations
Strong; the
convex-concave
interface
provides good
shape
complementarity
Unimportant
Successful
Variable, but
generally weak.
Charge-charge
interactions can
be strong
Generally
moderate
Unpredictable;
can be very
difficult, even
with know
hypervariable
regions of
antibody
Hits are found,
but are generally
lost in scoring
and ranking
Example
Trypsinogen and trypsin inhibitor (1cgi):
KD = 0.2 pM, DASA = 1950 Å2, and
DGdes = -18.3 kcal/mol. Most complexes of
enzymes with their protein inhibitors are
in this category
Ribonuclease a and ribonuclease
inhibitor (1dfj): DASA = 2580 Å2,
DGdes = 18.6 kcal/mol, DEelec=-63.9 kcal/mol
KD = 0.15 nM
Hyhel-5 Fab with lysozyme (1mlc):
KD = 126M, DASA = 1390 Å2, and DGdes = 3.84 kcal/mol. Most antibody – antigen
complexes are in this category
Ras and Ras interacting domain (1lfd)
KD = 2M, DASA = 1250 Å2, DGdes = 3.6
kcal/mol, and DEelec =-39.5 kcal/mol A
number of weak complexes are in this
category
Rigid body
Cyclin A and cyclin-dependent kinase 2
methods seem
(1fin): KD = 47.6 M, DASA = 3550 Å2,
to always fail for DGdes = 3.9 kcal/mol, and DEelec= -66.5
these complexes kcal/mol.
ASA – Acessible Surface Area, bStandard interface: 1400 Å2 < DASA < 2000 Å2, c C RMSD -  carbon Root Mean Square Deviation
ras-interacting domain
of ralgds
GNP
(5'-guanosyl-imido-triphosphate
ras protein
Interface in the complex of ras-interacting domain with ras
Desolvation free energy
Type IV
Difficult
Type III
Uncertain
Type II
Easy
Antibody/
Antigen
-4
Small
signalling
complexes
Type I
Easy
Large
multienzyme
complexes
Type II
Or
Type V?
Type V
Hopeless
Transitional
complexes with
substantial
conformational
change
Enzymes
1400
2000
3400
Interface Area
Classification of complexes
Type
a
Conformational
change
Interface
DASAa
Hydrophobicity
I
Small (rigid
interface)
Standardb
II
Small
DASA >
2000 Å2
III
Moderate, but
larger than for
Type I
Standard
IV
Restricted to
side chains
DASA <1400 Weak; mostly
polar and
Å2
charge-charge
interactions
V
Substantial
backbone
change,
C RMSD > 2 Å
DASA >
2000 Å2
Docking
outcome
Successful,
unless key side
chains are in
wrong
conformations
Strong; the
convex-concave
interface
provides good
shape
complementarity
Unimportant
Successful
Variable, but
generally weak.
Charge-charge
interactions can
be strong
Generally
moderate
Unpredictable;
can be very
difficult, even
with know
hypervariable
regions of
antibody
Hits are found,
but are generally
lost in scoring
and ranking
Example
Trypsinogen and trypsin inhibitor (1cgi):
KD = 0.2 pM, DASA = 1950 Å2, and
DGdes = -18.3 kcal/mol. Most complexes of
enzymes with their protein inhibitors are
in this category
Ribonuclease a and ribonuclease
inhibitor (1dfj): DASA = 2580 Å2,
DGdes = 18.6 kcal/mol, DEelec=-63.9 kcal/mol
KD = 0.15 nM
Hyhel-5 Fab with lysozyme (1mlc):
KD = 126M, DASA = 1390 Å2, and DGdes = 3.84 kcal/mol. Most antibody – antigen
complexes are in this category
Ras and Ras interacting domain (1lfd)
KD = 2M, DASA = 1130 Å2, and DGdes = 3.6
kcal/mol. A number of weak complexes
are in this category
Rigid body
Cyclin A and cyclin-dependent kinase 2
methods seem
(1fin): KD = 47.6 nM, DASA = 3550 Å2,
to always fail for DGdes = 3.9 kcal/mol, DEelec =-66.5 kcal/mol
these complexes
ASA – Acessible Surface Area, bStandard interface: 1400 Å2 < DASA < 2000 Å2, c C RMSD -  carbon Root Mean Square Deviation
Type V:
Large interface and
large conformational change
Cyclin-A
Cyclin-dependent kinase
Li, L. et al. (2003) RDOCK
Gray, J.J. et al. (2003)
2: How the community is doing?
Overall Success rates of participants in CAPRI 1-5
Classification of CAPRI 1-2 Targets
Desolvation free energy, kcal/mol
20
T7
15
Type III
uncertain
10
Type II
easy
Type IV
difficult
T5 T4 T3
5
0
Type V
very difficult
T6
T2 T1
-5
Type I
easy
-10
1000
1500
2000
2500
Interface area DASA
3000
3500
Overall Success rates of participants in CAPRI 1-5
Classification of CAPRI 3-5 Targets
Desolvation free energy, kcal/mol
25
20
Type III
uncertain
T10
15
T8
10
T19
5
0
T14
Type II
easy
T18
Type V
very difficult
T12
-5
T13
-10
Type I
easy
T9
-15
1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500
Interface area DASA
Overall Success rates of participants in CAPRI 1-5
Desolvation free energy
Type IV
Difficult
-4
Type III
Uncertain
Expected
Improvements
Type II
Easy
Small
Antibody/
signalling Antigen
complexes
Type I
Easy
Type II
Or
Type V?
Transitional
complexes with
substantial
conformational
change
Large
multienzyme
complexes
Much improved
Enzymes
1400
2000
3400
Interface Area
Type V
Hopeless
3. How to deal with side chain flexibility?
Fine details
Receptor
Ligand
Coarse details
Trypsin/APPI
Recognition mechanisms:
Lock-and-key vs. Induced fit
Key-and-latch mechanism
Rajamani, D., Thiel, S. Vajda, S. and C.J. Camacho. Anchor residues in protein-protein
interactions. Proc. Natl. Acad. Sci. USA, 101: 11287-11292, 2004.
Key-Latch model
key
latch
KEYS which stay close to the bound
conformation in solution
LATCHES do not show preference to stay near
bound conformation.
Individually crystallized protein
Predisposition
Unbound
Bound
Simulated
Solvated protein
RMSD of Arg39 of ribonuclease A with respect to the structure found in the
complex (bound; PDB code 1DFJ) and in the individually crystallized
ribonuclease A (unbound; PDB code 7RSA). The RMSD was computed for
2000 snapshots of a 4ns MD simulation of 7RSA.
7
Bound
Unbound
6
RMSD
5
4
3
2
1
0
1
2
Time (ns)
3
4
Clustering of the conformations of Arg39 in ribonuclease A. The 16 largest
clusters were derived from a pairwise RMSD analysis of the MD snapshots,
and clustering using a radius of 2Å. The RMSD of the cluster center from the
bound conformation is shown on the top/bottom of each bar. The bound
conformation is shown in blue, unbound in red, and the dominant conformation
from the MD simulations is shown in green.
Cluster size
300
200
2.4
1.5
1.8
2.4
2.2
100
2.7
3.3
0
2.3
2.9
2.4
1.8
1.7
Clusters
3.5
1.7
3.1
Complex of trypsin with amyloid β-protein inhibitor (APPI). Key residue
Arg-15 is a major contributor to the total binding free energy.
HIV-1 NEF/FYN tyrosine kinase SH3 domain complex. Trp-119 is within
1 and 2 Å of the bound conformation for 36% and 96% of the MD.
It is stabilized in this native-like conformation by Tyr-93 (and therefore also
native-like) in the free state. Thr-97 buries the second largest SASA (70 Å2).
Thr97
Tyr93
Asp100
Trp119
Hyhel-5 Fab/lysozyme complex. The main key residue, Arg-45, has a SASA
value of 147 Å2; a second key residue, Lys-68, is found buried with a
SASA = 93 Å2. Both side chains show native-like properties,
sampling during 50% and 97% of the time conformations that were less than
2 Å rmsd from their corresponding bound rotamer.
Lys68
Arg45
The complex of acetylcholinesterase with fasciculin. The main key Met-33 is in
a native-like conformation during most of the simulation. The SASA encompassed
by Met-33 is comparable with the next largest SASA of 78 Å2 resulting from the
burial of Arg-27; this anchor is in a native-like conformer during 95% of the MD.
Thr8
Arg27
Met33
ΔSASA
Complex
Receptor/Ligand
PDB ID
Anchora
b
ResID
Å2
ΔGbind
kcal/mol
Residence time, %c
Rank
MD
Rotamer
library
Enzyme/Inhibitor
1BRC
Trypsin/APPI (1AAP)
Arg 15
251.24
-11.9
1
32†
2SIC
Subtilisin BPN/Inhibitor
Met 70
196.33
-7.1
1
51†
2SNI
Subtilisin novo/CI2 (2CI2)
Ile 56
189.79
-7.6
1
37‡
1CHO
α-Chymotrypsin/OMTKY3
Leu 18
180.33
-7.9
1
73‡*
1CSE
Subtilisin C/eglin C (1ACB)
Leu 45
165.07
-5.1
1
50‡
1BRS
Barnase/barstar (1A19)
Asp 35
125.06
-2.5
3
97‡
1UGH**
UDG/UGI
Leu 272
180.38
-5.2
1
66‡
1DFJ
Ribonuclease inhibitor/
Asn 67
101.18
-1.2
8
41‡
7.4†
96.6‡
97.4‡
28.5‡
ribonuclease A (7RSA)
AchE/FasII (1FSC)
Thr 8
96.29
-3.4
4
99‡
1BQL
Hyhel5 Fab/QBL (1DKJ)
Arg 45
165.3
-10.1
1
49†
1FBI
IgG1 Fab/lysozyme
Arg 73
132.72
-1.9
4
46†
1DQJ
Hyhel63 Fab/HEL (3LZT)
Arg 21
131.4
5.4
1FSS
Antigen/Antibody
92†
38.3†
29.1†
Native-like Predictions
PDB
IDa
Receptor/Ligandb
Bound
c
UBd
e
Subtilisin Novo/Chymotrypsin
inhibitor2(2CI2)
RMSDBound
Predictions
UBg
MDh
Bi
MDj
Met59
2.9
1.5
99
82
Ile56
0.6
0.9
73
74
Residuef
Enzyme/Inhibitor complexes
2SNI
Anchor replacement
Resurf
151
62
151
62
124
1DFJ
Ribonuclease inhibitor/ Ribonuclease A
(7RSA)
63
22
43
Arg39
3.6
2.7
22
43
1BRC
Trypsin/APPI (1AAP)
98
24
45
Arg15
3.8
2.4
70
57
1CHO
A-Chymotrypsin/Ovomucoid 3rd domain
182
61
91
Leu/Met18
32
68
1BRS
Barnase/Barstar (1A19)
54
23
27
Asp35
0.8
0.6
16
17
1CSE
Subtilisin Carlsberg/Eglin C(1ACB)
176
105
116
Leu45
0.6
1
133
119
1FSS
Snake venom
acetylcholinesterase/FasciculinII (1FSC)
21
3
4
Thr9
0.5
0.6
4
5
2BTF
β-actin/Profilin
43
18
15
Arg74*
2.4
1.9
25
28
1WQ1
RAS activating domain/ RAS
29
26
9
Tyr32
0.2
1.5
26
21
Gln61*
2.4
1.5
28
40
Credits
Crystallographers: Please submit to CAPRI
Dr. Carlos Camacho (University of Pittsburgh)
Graduate students at Boston University
Stephen Comeau
Deepa Rajamani
Dima Kozakov
Yang Shen
Ryan Brenke
National Institute of Health