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the Modeling of Atomic Fluctuations in Protein Crystal Structures

Wenjun Zheng*

Physics Department, University at Buffalo, Buffalo, New York

ABSTRACT Protein conformational dynamics, despite its significant anharmonicity, has been widely explored by normal mode

analysis (NMA) based on atomic or coarse-grained potential functions. To account for the anharmonic aspects of protein

dynamics, this study proposes, and has performed, an anharmonic NMA (ANMA) based on the Ca-only elastic network models,

which assume elastic interactions between pairs of residues whose Ca atoms or heavy atoms are within a cutoff distance. The

key step of ANMA is to sample an anharmonic potential function along the directions of eigenvectors of the lowest normal modes

to determine the mean-squared fluctuations along these directions. ANMA was evaluated based on the modeling of anisotropic

displacement parameters (ADPs) from a list of 83 high-resolution protein crystal structures. Significant improvement was found

in the modeling of ADPs by ANMA compared with standard NMA. Further improvement in the modeling of ADPs is attained if the

interactions between a protein and its crystalline environment are taken into account. In addition, this study has determined the

optimal cutoff distances for ADP modeling based on elastic network models, and these agree well with the peaks of the statistical

distributions of distances between Ca atoms or heavy atoms derived from a large set of protein crystal structures.

INTRODUCTION

Conformational dynamics at the atomic level is increasingly oped to model protein dynamics at amino acid resolution.

recognized as important in protein functions including The ENM is usually constructed based on a Ca-only repre-

protein-ligand interactions, protein-protein interactions, and sentation of protein structures, where pairs of residues

allosteric regulation (13). Protein dynamics spans a wide whose Ca atoms or heavy atoms are within a cutoff distance

range of timescales, from femtoseconds to seconds. Of are connected by elastic springs with a uniform (12) or

particular interest is the long-time dynamics (microseconds distance-dependent (17,18) force constant. Such dramatic

to seconds) in large protein complexes, which is far beyond simplification allows the coarse-grained normal modes to

the timescales (nanoseconds to microseconds) of atomistic be calculated efficiently without energy minimization. Early

molecular dynamics (MD) simulations (4) despite fast- studies have shown that the large-scale collective motions

advancing computing technology (5). To capture such predicted by NMA of ENMs are insensitive to the dramatic

slow protein dynamics, normal mode analysis (NMA) was simplification in ENMs (13,14). Indeed, the lowest normal

developed and has been widely applied (610). In a typical modes calculated from ENMs were found to compare well

NMA of protein dynamics, an atomic or coarse-grained with many large-scale conformational changes observed by

potential function is approximated by a harmonic potential crystallography (13,19). Numerous studies have established

near a minimal-energy conformation, from which a Hessian ENM as an efficient means of probing the functionally

matrix (the second derivatives of potential function) is calcu- relevant protein dynamics with virtually no limit in timescale

lated. Then, a set of normal modes are solved from the or system size (2025).

Hessian matrix, and these can be used to describe small- Despite the great success of NMA, its underlying assump-

amplitude atomic motions at low temperatures. Under tion of harmonicity remains questionable. It is well known

harmonic assumption, the inverse of the eigenvalue of a that protein dynamics at physiological or lower temperatures

normal mode is proportional to the mean-squared fluctuation is highly anharmonic thanks to various factors, including

(MSF) of atomic coordinates along the direction of its solvation effect and the multiminima potential energy

eigenvector. function. For example, an early study using NMA and MD

To facilitate the application of NMA to large protein simulation found that an isolated bovine pancreatic trypsin

complexes, it is often performed based on a coarse-grained inhibitor (BPTI) exhibits marked anharmonic dynamics at

protein structural model (11). In recent studies, elastic temperatures of 100120 K (26). Therefore, the standard

network models (ENMs), including the anisotropic network NMA is quantitatively inaccurate to describe the anharmonic

model (ANM) (1214) and its isotropic variation, the atomic fluctuations in protein structures, which often results

Gaussian network model (GNM) (15,16), have been devel- in an underestimation of the MSF (27). To explore the anhar-

monic aspects of protein dynamics, the quasiharmonic

analysis was developed based on the principal component

Submitted January 5, 2010, and accepted for publication March 15, 2010. analysis of an MD trajectory (28). It yields quasiharmonic

*Correspondence: wjzheng@buffalo.edu modes that can be compared with the normal modes from

Editor: Gerhard Hummer. standard NMA. In one simulation study of BPTI, it was

! 2010 by the Biophysical Society

0006-3495/10/06/3025/10 $2.00 doi: 10.1016/j.bpj.2010.03.027

3026 Zheng

found that the first quasiharmonic mode shows barrier Based on the modeling of ADPs from a large set of 83

crossing events on an anharmonic energy surface, whereas high-resolution protein crystal structures studied previously

the higher quasiharmonic modes appear to be largely (39,41,43), the main findings are summarized as follows:

harmonic (27). In another simulation study of BPTI (29), it

1. ANMA significantly improves the modeling of aniso-

was shown that the larger the MSF of a quasiharmonic

tropic atomic fluctuations described by ADPs, especially

mode, the greater the degree of anharmonicity in its motion.

for low cutoff distances of ENMs. Unlike the standard

For BPTI, the anharmonic modes represent only 12% of the

NMA, ANMA is not susceptible to the tip effect, which

total number of variables, but they account for 98% of the

causes anomalously large fluctuations of some protruding

total MSF (29). Therefore, a proper account of anharmonic-

structural elements of a globular protein (45). Explicit

ity for the lowest quasiharmonic or normal modes is critical

accounting of the interactions between a protein and its

to accurate modeling of protein dynamics.

crystalline environment leads to further improvement in

Despite the strong effects of anharmonicity on the magni-

the modeling of ADPs.

tude of atomic fluctuations, it has been shown that the eigen-

2. By analyzing the cutoff-distance dependence of ADP

vectors of the lowest modes constitute an essential subspace

modeling, this work determines the optimal cutoff dis-

of collective coordinates, which offer good descriptions of

tances for three ENM schemes (ANMCa, ANMatom, and

collective motions in proteins (30,31). It is argued that the DNM; see Methods). The values of optimal cutoff

directions (but not the magnitude) of these collective distances correspond to a secondary peak in the distribu-

motions as captured by the eigenvectors of the lowest modes tion of Ca-Ca distances, and the upper bound of a broad

are insensitive to details of microscopic interactions and peak in the distribution of heavy-atom distances, which

solvent damping (25). Therefore, a promising recipe for are derived from a large set of protein crystal structures.

modeling anharmonicity within the framework of NMA is This finding hints at the dynamic importance of indirect

to keep the eigenvectors (but not eigenvalues) of standard atomic interactions beyond the range of direct interactions

NMA, and then sample atomic fluctuations/motions along via van der Waals or hydrogen-bond forces.

the directions of these eigenvectors using an anharmonic

potential function (see Methods). This general approach is

here referred to as anharmonic NMA (ANMA). A similar METHODS

strategy has been adopted in previous studies, where the ENM for an isolated protein structure

eigenvectors of lowest normal modes were utilized to

An ENM is constructed based on the Ca coordinates of a protein crystal

enhance MD simulations (32), refine x-ray diffraction data

structure. The potential energy of an ENM is

(33), and fit low-resolution electron microscopy maps (34)

and crystallographic B factors (35). 1X ! "2

E Cij dij " dij;0 ; (1)

This study employs ANMA to model anharmonicity 2 i<j

within the framework of ENM. The ENM potential function, where Cij is the force constant of the spring connecting Ca atoms i and j, dij is

despite its quadratic form (Eq. 1), is actually anharmonic in the atomic distance between Ca atoms i and j, and dij,0 is the value of dij

Cartesian coordinate space. This study will evaluate how given by the crystal structure. Three schemes of force constant assignments

ANMA improves the modeling of anisotropic atomic fluctu- are considered here:

ations obtained by x-ray crystallographic studies of protein 1. ANMCa:

structures. Atomic fluctuations in protein crystal structures #

C if dij;0 < Rc ;

have been traditionally quantified by the isotropic tempera- Cij ; (2)

0 otherwise

ture factors (or B factors), which use an isotropic Gaussian

where Rc is a cutoff distance that varies between 7 and 20 A, and the value of

distribution to characterize the spread of electron density constant C is determined by fitting the experimental ADPs (see below).

of each atom (36). Recently, a growing number of high-

2. ANMatom:

resolution protein crystal structures have been refined using X ! "

anisotropic Gaussian distributions, which characterize Cij C q Ratom " di1 j1 ;0 ; (3)

atomic fluctuations by a symmetric tensor with six indepen- i1 ;j1

dent elements called anisotropic displacement parameters where q () is the Heaviside function, and the summation is over all pairs of heavy

(ADPs) (37,38). Unlike the B factors, the ADPs describe atoms of residues i and j within a cutoff distance Ratom (Ratom varies between

not only the magnitude but also the direction of mean- 4 and 15 A). Here, i1 and j1 are indices for the heavy atoms of residues i and j.

squared atomic fluctuations. Therefore, they offer richer 3. DNM:

information of protein dynamics in crystalline states. ! "

X q Ratom " di1 j1 ;0

Modeling of ADPs based on NMA has been conducted in Cij C : (4)

i1 ;j1

di21 j1 ;0

several recent studies (3944), which only considered

harmonic contributions to protein dynamics. This study is, The DNM was proposed by Kondrashov et al. (39), who set force

to our knowledge, the first in which anharmonicity is consid- constants for several distance ranges to the reciprocal of the total number

ered in the NMA-based modeling of ADPs. of atomic contacts in each range. Because the number of atomic contacts

Anharmonic Normal Mode Analysis 3027

grows quadratically with distance (assuming the atomic density is constant), protein structure (corresponding to an asymmetric unit of a protein crystal)

this works DNM formulation is essentially a continuous counterpart of the and an environment that includes the Ca atoms of neighboring protein mole-

original DNM (39). cules in a crystal. To reduce system size, the environment is truncated by

The Hessian matrix, H, is calculated as the second derivative of potential keeping the Ca atoms of neighboring molecules within 25 A from the Ca

energy E (see Eq. 1) with respect to the Cartesian coordinates of Ca atoms. atoms of the main protein structure (43). The Ca coordinates of such a trun-

For a protein structure with N residues, H contains N # N superelements cated environment are generated using the What If webserver (http://swift.

(called Hij) with size 3 # 3: cmbi.ru.nl/servers/html/index.html). To reduce the computing cost, one

2 3 assumes that only the Ca atoms of the main protein structure can move,

v2 E v2 E v2 E whereas those of the environment are fixed (43).

The potential energy of the two-component ENM is

6 vxi vxj vxi vyj vxi vzj 7

Hij 6 v2 E v2 E v2 E

; 7 (5) E

1X ! "2 1

Cij dij " dij;0 fenv

X 2

CiI diI " diI;0 ; (7)

4 vyi vxj vyi vyj vyi vzj 5 2 i<j 2 i;I

v2 E v2 E v2 E

where i and j (I) are indices for Ca atoms in the main structure (environment),

vzi vxj vzi vyj vzi vzj Cij (CiI ) is the force constant of the spring connecting Ca atoms i and j (I),

where xi,yi, and zi are the Cartesian coordinates of the Ca atom i. dij(diI) is the atomic distance between Ca atoms i and j (I), and dij,0 (diI,0)

is the value of dij given by the crystal structure. A parameter fenv within

A subset of total 3N normal modes is then solved. For each mode m,

the range [0, 1] is introduced to tune the strength of protein-environment

its eigenvalue (represented by lm ) and eigenvector (represented by Vm) are

interactions. fenv 0 corresponds to the case of an isolated protein structure

obtained by solving HVm lm Vm .

(see Eq. 1). fenv 1 if one assumes equal strength of interprotein and intra-

protein interactions. As shown in a recent work, a small fenv (%0.05) should

Anharmonicity of the ENM potential function

be used to accurately model protein dynamics in crystalline states (43).

Although the ENM potential function in Eq. 1 has a quadratic form, it is A small fenv implies significantly weaker interactions between neighboring

actually anharmonic in terms of Cartesian coordinates. In fact, the NMA proteins than within a protein (for details, see Hafner and Zheng (43)).

of ENM is based on the Taylor expansion of Eq. 1 truncated at the second

order, with higher-order anharmonic terms ignored. Let us consider the

Calculation of ADPs and B factors using ANMA

elastic interaction energy between Ca atoms i and j after they undergo

three-dimensional displacements D~ r j from their equilibrium posi-

r i and D~ and NMA

tions. This energy is expanded to the fourth order as follows (for a detailed Based on NMA, the covariance matrix of Ca coordinates of the main protein

derivation, see Supporting Material): structure can be constructed as

1

! "2 % & X

C

2 ij

dij " dij;0 uuT wm Vm VmT ; (8)

q

h !

"2 i2

m

12Cij dij;0 Drij;L Drij;T 2

" dij;0

h (6) where wm gives the magnitude of MSF along the direction of eigenvector Vm

2

Cij dij;0 4 of mode m, and the summation is over a subset of lowest normal modes (see

% 2

a2 ab2 b4 " a2 b2 i below). Under the harmonic assumption of NMA, wm kBT/lm , where kB is

fifth " or higher " order terms ; the Boltzmann constant and T is temperature. In ANMA, wm is determined

by uniformly sampling the ENM potential function (see Eqs. 1 and 7) along

where the relative displacement D~ r j " D~ r i is partitioned into a longitudinal the direction of Vm:

component, Drij;L D~ rj " D~ ri $~ nij0 , and a transverse component,

q L '

P

Drij;T jD~ r j " D~ ri j2 " Drij;L 2 , and ~ nij0 is the unit vector pointing from 'DXml j2 e"EDXml =kB T

l "L

the equilibrium position of Ca atom i to that of Ca atom j, a Drij;L =dij;0 , wm ; (9)

and b Drij;T =dij;0 . If the Taylor expansion in Eq. 6 is truncated at the P

L

e"EDXml =kB T

second order, only the longitudinal component, Drij;L , contributes, whereas l "L

the transverse component, Drij;T , does not. Therefore, a proper modeling of

transverse fluctuations between pairs of Ca atoms requires third- or higher- where DXml lAm =LVm is the lth displacement vector of Ca atoms along

order terms of Eq. 6. This cannot be achieved in standard NMA, which the direction of Vm, the maximal magnitude Am is chosen such that

ignores these anharmonic terms. To evaluate the effect of anharmonic terms EDXmL R5kB T, and L is the number of sample displacements in the direc-

on atomic fluctuations, I sample along the direction of the eigenvector of tion of Vm(or -Vm). To ensure good convergence, L is set to 20.

each mode using the exact form of ENM potential energy (Eq. 1). For an isolated protein, the potential energy function of Eq. 1 is used to

To demonstrate the anharmonicity of ENM potential energy, this study sample along the directions of eigenvectors of a selected subset of normal

has shown the ENM energy profiles for the displacements along the eigen- modes, which include the lowest 5% of modes with positive eigenvalue,

vectors of the lowest 10 modes, which are solved for an Escherichia coli and nontranslation/nonrotation zero modes, if present. The nontranslation/

hppk crystal structure (using ANMCa with Rc10 A) (see Fig. S1 in the Sup- nonrotation zero modes often result from the low connectivity of a subset

porting Material). Significant deviation from a harmonic potential is indeed of residues to the rest of the ENM, which tends to occur when a relatively

observed, accompanied by slight asymmetry between positive and negative small cutoff distance, Rc, of ENM is used. These zero modes cannot be prop-

displacements (Fig. S1). erly considered in standard NMA because of their anomalous contributions

to atomic fluctuations. The translation/rotation zero modes are excluded

ENM for a protein structure interacting because their contributions to atomic fluctuations are anomalous for an iso-

with a crystalline environment lated protein. For a protein interacting with its environment, the potential

energy function of Eq. 7 (with fenv 0.05) is used to sample along the direc-

To model the effects of crystal packing on protein dynamics, I construct tions of eigenvectors of a selected subset of normal modes, which includes

a Ca-only ENM consisting of two components: the Ca atoms of a main the lowest 5% of modes with positive eigenvalue, and all zero modes

3028 Zheng

(including six translation/rotation modes). The translation/rotation modes (dak and dbk, k 1,2,3) and eigenvectors (vakand vbk, k 1,2,3) of U and

can be included because their contributions to atomic fluctuations are no V as follows:

longer anomalous in the presence of protein-environment interactions.

The anharmonicity of mode m is assessed by the parameter- 3 1 X 3

dbk 1X 3 X 3

dak T 2

AHm lm wm =kB T(AHm< 1 if the anharmonicity reduces the MSF along Dab " ln jv vbl j : (14)

2 2 k 1 dak 2 k 1 l 1 dbl ak

mode m; AHm> 1 if the anharmonicity increases the MSF along mode m).

It is noted that AHm 1 does not necessarily mean perfect harmonicity. Since the KL distance is asymmetrical Dab sDba , the arithmetic average

AHm values are averaged over the above selected subset of normal modes Dab Dba =2 was calculated (40). Note that Dab diverges if the distribution

to assess their overall anharmonicity. b is highly anisotropic (with a near-zero eigenvalue). To avoid such diver-

The ith 3 # 3 diagonal block of the covariance matrixhuuT i gives the theo- gence, this study uses minfDab ; Dba g instead of Dab Dba =2 as this

retical prediction of the ADP tensor for Ca atom i: studys KL distance metric (43).

2 % & 3 2 3

dxi2

hdxi dyi i hdxi dzi i U11 U12 U13 Pearson correlations

% T& 6 % & 7 6 7

uu ii 4 hdxi dyi i dy2i hdyi dzi i5 4 U12 U22 U23 5; The above two metrics evaluate only the directional similarity of two ADPs.

% 2&

hdxi dzi i hdyi dzi i dzi U13 U23 U33 To include the magnitude of ADPs in the comparison, this study computes

0

the Pearson correlation (PCall) between two sets of ADPs as two 6N -dimen-

(10) ~ and V(40)

sional vectors U ~ 0

(N is the number of ADPs):

where the diagonal elements U11, U22, and U33 give the MSF of Ca atom i

6N 0 !

P "! "

along the x, y, and z directions, and the off-diagonal elements U12, U13, and U ~ V

~ j " hUi ~

~ j " hVi

U23 describe the covariance among the displacements of Ca atom i along the j1

x, y, and z directions. Together, the six ADP elements determine a three- PC s: (15)

P6N 0 ! " P 6N 0 ! "

dimensional Gaussian distribution function that describes both the direction ~ j " hUi ~ 2 ~ j " hVi ~ 2

and magnitude of the atomic fluctuations (38). For fixed probability value,

U V

j1 j1

the distribution is ellipsoidal, with a directional preference along the long

axis, which is given by the eigenvector of the ADP tensor with the largest 0

The Pearson correlations is also calculated for 3N diagonal and 3N

0

eigenvalue. The anisotropy of the Gaussian distribution is defined as the off-diagonal ADP elements separately (PCdiagonal and PCoffdiagonal, respec-

ratio of the smallest to the largest eigenvalue of the ADP tensor. tively), as well as the Pearson correlation between theoretical and experi-

The B factor is related to the trace of the ADP tensor as mental B factors (PCB) (40).

B 8p2 U11 U22 U33 =3: (11)

Crystallographic dataset for model evaluation

The modeling of ADPs is evaluated using a set of 83 high-resolution crystal

Comparison between theoretical structures previously studied in Kondrashov et al. (39). From the ANISOU

and experimental ADPs records of these PDB structures, 16,852 usable ADPs are collected for those

Ca atoms with occupancy of 1.0 (though all Ca atoms are included in the

To fit the theoretical ADPs (Eq. 10) to experimental ADPs for a protein

construction of ENM).

structure with N Ca atoms (one ADP tensor per Ca atom), the force constant

Following Kondrashov et al. (39), for the evaluation of four Pearson

parameter, C (see Eqs. 24), is adjusted so that the sum of 3N diagonal

correlations, all 16,852 ADPs are used; for the evaluation of two directional

elements matches up between the theoretical and experimental ADPs. Three

metrics (ccmod and KL distance), a subset of 6784 ADPs with aniso-

metrics are used to assess the similarity between two ADP tensors (repre-

tropy %0.5 are used.

sented as U and V).

Real-space correlation coefficient Evaluation of the statistical significance

The real-space correlation coefficient is calculated to evaluate the overlap inte- of improvement in ADP modeling

gral of two three-dimensional Gaussian distributions given by U and V (38): The ADP similarity metrics defined above are calculated for 6784 ADPs from

! "

"1 1=4

83 PDB structures using both NMA and ANMA. For a given metric S, the

detU "1 detV difference between NMA and ANMA, SANMA - SNMA, is calculated for

ccU; V ( )1=2 : (12)

each usable ADP (if S is ccmod or KL distance) or PDB structure (if S is PCall,

detU "1 V "1 =8

PCB, PCdiagonal, or PCoffdiagonal). To evaluate the statistical significance of this

Based on the real-space correlation coefficient, the modified correlation coef- difference, its average hSANMA " SNMA i and standard deviation sdS over

ficient (ccmod) is introduced to evaluate the directional similarity of two ADPs: 6784 usable ADPs or 83 PDB structures is calculated. Then, a Z score for S is

defined as

ccU; V " ccU; V ( ! p"

ccmod U; V ; (13) ZS hSANMA " SNMA i= sdS = Nsamp ; (16)

1 " ccU; V (

where Nsamp 6784 if S is ccmod or KL distance, and Nsamp 83 if S is PCall,

where V* is a 3 # 3 tensor generated by taking the eigenvectors of U and

PCB, PCdiagonal, or PCoffdiagonal. (For KL distance, a minus sign is added to

using the eigenvalues of V, with the largest and smallest switched, to define

Eq. 16 to make ZS positive). A large Z score indicates high statistical signif-

the two ellipsoids with perfect misalignment (39). ccmod is 1.0 (0) if the two

icance for the improvement in ADP modeling from NMA to ANMA.

ellipsoids are perfectly aligned (misaligned).

Kullback-Leibler distance

RESULTS

The Kullback-Leibler (KL) distance (46) evaluates the difference between

the three-dimensional Gaussian distributions a and b as defined by U and To explore how the anharmonicity of the potential energy

V (40). The KL distance can be expressed in terms of the eigenvalues function affects NMA-based modeling of atomic fluctuations

Anharmonic Normal Mode Analysis 3029

in protein crystal structures, this study has proposed and per- vector of each mode, and evaluate the MSF along these

formed an anharmonic NMA (ANMA) in the absence and directions; and 3), I construct a covariance matrix to calcu-

presence of interactions between a protein and its crystalline late the ADP tensors. As a control, ADP tensors using stan-

environment. This study focuses on the anharmonicity of dard NMA (43) are calculated. The theoretical ADPs are

ENM potential energy function. ANMA has been systemat- compared with experimental ADPs using various metrics

ically compared with standard NMA based on the modeling to assess the modeling quality (see Methods). To ensure

of a large set of ADPs from 83 high-resolution protein crystal the robustness of the modeling results, I consider three

structures (39). This work now presents the findings based ENM schemes: ANMCa, ANMatom, and DNM (see Methods).

on the modeling results. As an example, this work shows the results of ADP

modeling using ANMCa (with Rc 7 A) for an E. coli 6-hy-

droxymethyl-7,8-dihydropterin pyrophosphokinase (hppk)

ANMA improves ENM-based ADP modeling

crystal structure (PDB code: 1F9Y) (Fig. 1 a). ANMA gives

In previous ENM-based studies of atomic fluctuations in better agreement between theoretical and experimental

protein crystal structures (17,3941,43,44,47,48), it was ADPs than does NMAthe Pearson correlations for all,

implicitly assumed that the atomic fluctuations are harmonic. diagonal, and off-diagonal ADP elements and for B factors

Thus, the MSF along the direction of the eigenvecgtor of a (PCall, PCdiagonal, PCoffdiagonal, PCB; see Methods) increase

normal mode is proportional to the inverse of its eigenvalue. from 0.27, 0.18, 0.06, and 0.25 to 0.58, 0.48, 0.30, and

This is not a realistic assumption for protein dynamics at 0.60, respectively. For the directional comparison of experi-

physiological temperature or crystallographic temperature mental and theoretical ADPs, this study focuses on 85 of

(26). There are two main sources of anharmonicity in protein 158 experimental ADPs with anisotropy %0.5 (41). Two

dynamics: the presence of multiple minima in a potential metrics for directional similarity between theoretical and

energy function and the deviation of a single-minimum experimental ADPs (ccmod and KL distance; see Methods)

potential function from a harmonic well. Many previous are calculated, which also indicate improvement from

studies have addressed the former case using MD simula- NMA to ANMAthe average ccmod increases from 0.33

tions and quasiharmonic analysis (27,29). Here, the latter to 0.43 (Fig. 1 c) and the average KL distance decreases

case is addressed. In particular, this study explores how the from 0.91 to 0.58 (Fig. 1 d). Compared with NMA,

anharmonicity of the ENM potential function affects ANMA has suppressed two pronounced peaks in the theoret-

ENM-based modeling of atomic fluctuations in protein ical B factors (Fig. 1 b) and KL distances (Fig. 1 d), which

crystal structures. To this end, this study proposes and correspond to two floppy loops (residues 4550 and 8590)

evaluates the ANMA protocol (for details, see Methods): (Fig. 1 a). The account of anharmonicity by ANMA im-

1), I solve a subset of lowest normal modes for an isolated proves the modeling of atomic fluctuations in these dangling

protein structure modeled by ENM; 2), I sample the ENM loops, as suggested by the higher ccmod (Fig. 1 c) and lower

potential energy function along the direction of the eigen- KL distance (Fig. 1 d).

E. Coli hppk crystal structure (PDB code: 1F9Y).

(a) A cartoon representation of 1F9Y with two

floppy loops (residues 4550 and 8590) colored

black. (b) B factors (rescaled by 3/8p2) from crys-

tallography (dotted line), modeled by NMA (gray

line) and by ANMA with fenv 0 (black line)

(the positions of the two loops shown in a are

underscored). (c and d) ccmod and KL distance,

respectively, for NMA (gray lines) and ANMA

with fenv 0 (black lines). (e) B factors (rescaled

by 3/8p2) from crystallography (dotted line),

modeled by ANMA with fenv 0 (gray line) and

by ANMA with fenv 0:05 (black line). (f and g)

ccmod and KL distance, respectively, for ANMA

with fenv 0 (gray lines) and ANMA with

fenv 0:05 (black lines).

3030 Zheng

ADPs collected from 83 protein crystal structures (39). To

assess the performance of ADP modeling, this study aver-

ages four Pearson correlations (PCall, PCB, PCdiagonal, and

PCoffdiagonal) over 83 structures, and two directional metrics

(ccmod and KL distance) over a subset of 6784 ADPs with

anisotropy %0.5. These average metrics are calculated as

a function of cutoff distance for ANMA and NMA based

on three ENM schemes (ANMCa, ANMatom, and DNM;

see Methods). The results are shown in Figs. 24 and Tables

S1S3. Z scores for the above metrics are calculated to assess

the statistical significance of the improvement from NMA to

ANMA (see Methods).

For ANMCa, significant improvement was found in ADP

modeling from NMA to ANMA, especially for Rc< 10 A

(see Table S1 in the Supporting Material and Fig. 2, a and b).

For example, for Rc 7 A, the average Pearson correlations

(PCall, PCdiagonal, PCoffdiagonal, and PCB) increase from 0.31,

0.22, 0.14, and 0.28 to 0.54, 0.39, 0.26, and 0.47; the average FIGURE 3 The results of ADPs modeling averaged over 83 protein

ccmod increases from 0.45 to 0.50; and the average KL crystal structures (39) using ANMatom and (a) NMA; (b) ANMA

withfenv 0; (c) ANMA with fenv 0:05. Shown here are Pearson correla-

distance decreases from 0.76 to 0.48. These improvements

tions of diagonal (9), off-diagonal (8), and all elements (7) of ADPs, and

are highly significant, as indicated by large Z scores >10 B factors (6), and directional metrics including ccmod (B) and KL distance

(Table S1). For Rc R 10 A, the improvement from NMA (,) as a function of cutoff distanceRatom . Also shown in b is the average

to ANMA is smaller. For example, for Rc 10 A, the anharmonicity, AH (>).

average Pearson correlations (PCall, PCdiagonal, PCoffdiagonal,

and PCB) increase from 0.43, 0.36, 0.24, and 0.43 to 0.48, For ANMatom and DNM, significant improvement was

0.40, 0.28, and 0.47; the average ccmod increases from also found in ADP modeling from NMA to ANMA, espe-

0.50 to 0.52; and the average KL distance decreases from cially at the low cutoff distance Ratom < 8 A (for details,

0.48 to 0.43. This smaller improvement remains statistically see Table S2, Table S3, and Figs. 3, a and b, and 4, a and b).

significant, as the Z scores for all metrics are >5 (Table S1). Therefore, the finding that ANMA improves ENM-based

ADP modeling is robust and does not rely on details of

ENM schemes.

An ENM with low cutoff distance is prone to the so-called

tip effect, which involves some pathological modes

describing anomalously large motions of some protruding

structural elements (such as a dangling loop) while the rest

of the protein remains essentially static (45). The large local

motions in these modes dramatically reduce the accuracy of

harmonic approximation to the ENM potential function (see

Eq. 6), therefore making those modes highly anharmonic.

Indeed, for ANMCa (ANMatom and DNM) the average

anharmonicity AH (see Methods) decreases sharply as

Rc(Ratom) drops below 10 A (8 A) (see Figs. 2 b, 3 b, and

4 b). Therefore, it is not surprising that the proper account

of anharmonicity by ANMA significantly improves ADP

modeling at low cutoff distance. Thus, an accurate modeling

of protein atomic fluctuations demands a proper account of

the anharmonicity of the ENM potential function.

FIGURE 2 The results of ADP modeling averaged over 83 protein crystal

structures (39) using ANMCa and (a) NMA, (b) ANMA with fenv 0, and further improves ADP modeling by ANMA

(c) ANMA with fenv 0:05. Shown here are Pearson correlations of diag-

Previous studies have shown the significant effect of crystal-

onal (9), off-diagonal (8), all elements (z.7) of ADPs and B factors

(z.6), and directional metrics including ccmod (B) and KL distance (,) line environment on protein dynamics in crystalline states

as a function of cutoff distance Rc . Also shown in (b) is the average anhar- (41,43,47,48). In particular, the incorporation of protein-

monicity AH (>). environment interactions allows proper modeling of the

Anharmonic Normal Mode Analysis 3031

(39) are shown in Figs. 2 c, 3 c, and 4 c, and Table S1, Table S2,

and Table S3, which are compared with the results for

isolated protein structures (see Figs. 2 b, 3 b, and 4 b,

and Table S1, Table S2, and Table S3). The findings are

summarized as follows.

For ANMCa, a significant improvement was found in

modeling of the direction of ADP tensors with the addition

of protein-environment interactions (Fig. 2, b and c)for

example, for Rc 10 A, the average ccmod increases from

0.52 to 0.70 and the average KL distance decreases from

0.43 to 0.14. Furthermore, there is large improvement in

the modeling of both magnitude and direction of ADP

tensors as assessed by the four Pearson correlations.

For ANMatom and DNM, a significant improvement was

also found in the modeling of ADP tensors (see Figs. 3,

b and c, and 4, b and c). Therefore, this finding is robust

and does not rely on details of ENM schemes.

crystal structures (39) using DNM and (a) NMA; (b) ANMA with Optimal cutoff distances of ENM for ADP modeling

fenv 0; (c) ANMA with fenv 0:05. Shown here are Pearson correlations by ANMA

of diagonal (9), off-diagonal (8), all elements (7) of ADPs and B factors

(6), and directional metrics including ccmod (B) and KL distance (,) as In previous studies, it was found that the optimal fitting of

a function of cutoff distanceRatom . Also shown in b is the average anharmo- B-factors by ANMCa is attained at a high cutoff distance

nicity, AH (>). Rc 1524 A (49), which is beyond the range of Ca-Ca

distances between contacting residues (4.412.8 A (50)).

contributions of rigid-body motions of an entire protein to Some recent studies have addressed this inconsistency in

ADPs (41,43,48), which cannot be easily attained for an iso- ENM parameterization (43,51). To further address this issue,

lated protein structure. Therefore, it will be interesting to this study evaluates the quality of ADP modeling by NMA and

incorporate protein-environment interactions in the ANMA ANMA as a function of cutoff distance using three ENM

formulation in this study. To this end, a two-component schemes (Rc for ANMCa, Ratom for ANMatom, and DNM).

ENM comprised of a protein structure and its fixed crystalline The Rc dependence for ANMCa is summarized in Fig. 2.

environment has been constructed (see Methods). I have For NMA (Fig. 2 a), the Rc dependence of various ADP

recently found the interactions between a protein and its crys- comparison metrics diverges: PCdiagonal, PCoffdiagonal, and

talline environment to be much weaker than the intraprotein PCB saturate as Rc increases from 9 to 20 A, whereas PCall

interactions (43). ANMA was performed as follows to peaks at Rc ~ 9 A; ccmod peaks at Rc ~ 10 A; and the KL

account for the protein-environment interactions: first, solve distance is minimal at Rc ~ 10 A. Such divergence was

a subset of lowest normal modes for an isolated protein struc- also found in a previous study (43), which makes it hard to

ture; then sample the ENM potential energy function (with the parameterize ENM by optimizing the fitting of ADPs.

protein-environment interactions included; see Eq. 7) along For ANMA (Fig. 2 b), the divergence of the Rc dependence

the direction of the eigenvector of each mode; then calculate of various ADP comparison metrics is reduced: PCdiagonal,

the ADPs based on the sampling results (see Methods). PCoffdiagonal, PCB and PCall peak at Rc ~ 8 A, ccmod peaks at

As an example, this study shows and compares the results Rc ~ 9 A, and the KL distance is minimal at Rc ~ 9 A. Thus,

of ADP modeling by ANMA in the absence and presence a consensus-based optimization of various ADP comparison

of protein-environment interactions (using ANMCa with metrics points to an optimal Rc of ~89 A, which falls well

Rc 7 A) for an E. Coli hppk crystal structure (PDB within the range of Ca-Ca distances between contacting resi-

code: 1F9Y) (Fig. 1 a). Large improvement in ADP dues (4.412.8 A (50)). Note that the addition of protein-envi-

modeling is found in terms of the four Pearson correla- ronment interactions does not change the optimal Rc values

tionsPCall, PCdiagonal, PCoffdiagonal, and PCB change from (Fig. 2 c).

0.58, 0.48, 0.30, and 0.60 to 0.75, 0.56, 0.39, and 0.65. The Ratom dependence for ANMatom and DNM is summa-

Furthermore, improvement is found in terms of the direc- rized in Figs. 3 and 4, respectively. For ANMA based on

tional similarity in ADPsthe average ccmod increases ANMatom (Fig. 3 b), the Rc dependence differs among

from 0.43 to 0.66 (Fig. 1 f), and the average KL distance various ADP comparison metrics: PCdiagonal, PCall and PCB

decreases from 0.58 to 0.18 (Fig. 1 g). peak at Ratom ~ 5 A, PCoffdiagonal peaks at Ratom ~ 7 A,

The results of ADP modeling in the presence of protein- whereas ccmod peaks at Ratom ~ 8 A, and the KL distance

environment interactions for 83 protein crystal structures is minimal at Ratom ~ 8 A. The optimal Ratom values are

3032 Zheng

ment interactions (Fig. 3 c).

For ANMA based on DNM, the results are similar. For

example, ccmod peaks at Ratom ~ 9 A, and the KL distance

is minimal at Ratom ~ 9 A (Fig. 4 b), which agrees with the

results of Riccardi et al. (41). The optimal Ratomvalues are

only slightly shifted with the addition of protein-environ-

ment interactions (Fig. 4 c).

This works finding that the optimal modeling of the direc-

tion of ADPs by ANMatom (DNM) is at Ratom ~ 8 A (9 A) is

surprising, because this heavy-atom distance is beyond the

range of van der Waals interactions (<4 A as estimated

based on van der Waals radii (52)) or hydrogen-bond inter-

actions (<4.5 A as defined in the CONGEN program (53)).

with optimal cutoff distances

To rationalize the finding of an optimal cutoff distance of

~10 A for ANMCa, this work analyzes the statistical distribu-

tion, r(r), of distances between the Ca atoms of nonbonded

residues of 83 protein crystal structures (39). r(r) is given by

f(r)/4pr2, where f(r) is the percentage of all Ca-Ca distances

within the range (r " 0.5, r 0.5), and r is sampled at integer

values (in A). It is interesting that this study found that r(r)

has a primary peak around 56 A and a secondary peak at

10 A (Fig. 5 a). These two peaks correspond to two minima

of a statistical potential function based on Ca-Ca distances.

The coincidence of the secondary peak of r(r) and the

optimal Rc for ANMCa hints at the presence of effective FIGURE 5 The density functions for the statistical distributions of

interactions between residues separated by a Ca-Ca distance distances between (a) Ca atoms and (b) heavy atoms of nonbonded residues

in 83 protein crystal structures (39). r(r) is rescaled by the density function

of %10 A, which is important to protein dynamics. r0 for a uniform distribution. The same statistical distributions (not shown

In a similar way, to rationalize the finding of an optimal here) are obtained from a much larger set of >2000 protein crystal structures

atomic distance of ~8 A (9 A) for ANMatom (DNM), this (cullpdb_pc30_res1.6_R0.25_d081223_chains2039, downloaded from

work analyzes the statistical distribution of distances between http://dunbrack.fccc.edu/PISCES.php).

the heavy atoms of nonbonded residues of 83 protein crystal

structures (39). The distribution shows a broad peak ranging

from 5 to 10 A (Fig. 5 b), which suggests the presence of statis- 83 high-resolution protein crystal structures. Significant

tical interactions between heavy atoms from different residues improvement was found in the modeling of ADPs by

separated by %10 A. This value is beyond the range of van der ANMA compared with standard NMA. Further improve-

Waals or hydrogen-bond interactions. The idea that long-range ment in the modeling of ADPs is attained if the interactions

electrostatic interactions may be the cause is ruled out, because between a protein and its crystalline environment are taken

the same distribution is obtained after charged residues are into account. In addition, this work has determined the

excluded from the statistical analysis. This observation roughly optimal cutoff distances for ENM-based ADP modeling,

agrees with this studys finding of Ratom ~ 8 A (9 A) as the which agree well with the peaks of the statistical distribu-

optimal atomic distance for ANMatom (DNM). Taken together, tions of distances between Ca atoms or heavy atoms deduced

these findings support the presence of indirect interactions from a large set of protein crystal structures.

between atoms separated by %10 A and the importance of This work applied ANMA to explore the anharmonicity of

these interactions to protein dynamics. Such effective interac- the ENM potential function (Eq. 1). It is straightforward to

tions may arise as a result of averaging over some hidden vari- apply ANMA to any other anharmonic potential function

ables (such as multiple substates of a protein native state). (such as a Lennard-Jones potential). In fact, I applied

ANMA to the generalized potential function

2

En dij 0:5Cij dij;0 n

=n2 1 " dij;0 =dijn 2 , which is reduced to

DISCUSSION AND CONCLUSION

the ENM potential in Eq. 1 when n "1 and to a Len-

To account for the anharmonic aspects of protein dynamics, nard-Jones potential when n 6. The application of

this study has used ANMA to model the ADPs from a set of ANMA to this generalized potential function, however,

Anharmonic Normal Mode Analysis 3033

does not lead to further improvement of ADP modeling (data 11. Tozzini, V. 2005. Coarse-grained models for proteins. Curr. Opin.

Struct. Biol. 15:144150.

not shown).

12. Tirion, M. M. 1996. Large amplitude elastic motions in proteins from

In this study, the anharmonicity, AH, is found to be <1

a single-parameter, atomic analysis. Phys. Rev. Lett. 77:19051908.

(see Figs. 2 b, 3 b, and 4 b). Therefore, anharmonicity of

13. Tama, F., and Y. H. Sanejouand. 2001. Conformational change of

the ENM potential function results in the stiffening of a proteins arising from normal mode calculations. Protein Eng. 14:16.

normal mode (or reduction of the MSF along the direction 14. Atilgan, A. R., S. R. Durell, ., I. Bahar. 2001. Anisotropy of fluctua-

of its eigenvector). This finding complements the early find- tion dynamics of proteins with an elastic network model. Biophys. J.

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multiple potential energy minima (27,29). 15. Bahar, I., A. R. Atilgan, and B. Erman. 1997. Direct evaluation of

thermal fluctuations in proteins using a single-parameter harmonic

By using a fixed set of eigenvectors solved by standard potential. Fold. Des. 2:173181.

NMA, this study focuses on the anharmonic effect on the 16. Haliloglu, T., I. Bahar, and B. Erman. 1997. Gaussian dynamics of

magnitude of atomic fluctuations. It will be interesting to folded proteins. Phys. Rev. Lett. 79:30903093.

explore in future studies the anharmonic effect on the direc- 17. Kondrashov, D. A., Q. Cui, and G. N. Phillips, Jr. 2006. Optimization

tion of atomic fluctuations. and evaluation of a coarse-grained model of protein motion using x-ray

This study offers a new avenue to the accurate parameter- crystal data. Biophys. J. 91:27602767.

ization of ENM beyond the limit of harmonic approximation. 18. Yang, L., G. Song, and R. L. Jernigan. 2009. Protein elastic network

models and the ranges of cooperativity. Proc. Natl. Acad. Sci. USA.

The correlation of optimal ENM parameters with statistical 106:1234712352.

distributions of atomic distances may also offer a systematic 19. Krebs, W. G., V. Alexandrov, ., M. Gerstein. 2002. Normal mode

way to develop ENM parameters based on residue type. analysis of macromolecular motions in a database framework: devel-

A more accurate ENM is essential to the development and oping mode concentration as a useful classifying statistic. Proteins.

48:682695.

refinement of ENM-based techniques that probe protein

20. Bahar, I., and A. J. Rader. 2005. Coarse-grained normal mode analysis

dynamics of functional relevance (5457). in structural biology. Curr. Opin. Struct. Biol. 15:586592.

21. Tama, F., and C. L. Brooks. 2006. Symmetry, form, and shape: guiding

principles for robustness in macromolecular machines. Annu. Rev.

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One figure and three tables are available at http://www.biophysj.org/ 22. Rader, A. J., D. H. Vlad, and I. Bahar. 2005. Maturation dynamics of

biophysj/supplemental/S0006-3495(10)00359-0. bacteriophage HK97 capsid. Structure. 13:413421.

23. Wang, Y., A. J. Rader, ., R. L. Jernigan. 2004. Global ribosome

The author thanks the American Heart Association (0835292N) for funding motions revealed with elastic network model. J. Struct. Biol.

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24. Lezon, T. R., A. Sali, and I. Bahar. 2009. Global motions of the nuclear

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