obstruction of dengue virus maturation by fab fragments...

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Obstruction of Dengue Virus Maturation by Fab 1

Fragments of the 2H2 Antibody 2

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Zhiqing Wanga, Long Lia,b, Janice G. Penningtona,c, Ju Shenga, Moh-Lan Yapa, Pavel 4

Plevkaa, Geng Menga, Lei Suna, Wen Jianga, and Michael G. Rossmanna,# 5

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aDepartment of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West 7

Lafayette, IN 47907 8

bPresent address: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave, 9

Boston, MA 02115 10

cPresent address: Department of Botany, 430 Lincoln Drive, University of Wisconsin-Madison, 11

Madison, WI 53706 12

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Corresponding author: [email protected] 14

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Running Title: Immature DENV complexed with 2H2 Fab 17

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Abstract Word Count: 234 19

Text Word Count: 2848 20

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00472-13 JVI Accepts, published online ahead of print on 5 June 2013

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ABSTRACT 22

The 2H2 monoclonal antibody recognizes the precursor peptide on the immature dengue 23

virus and might, therefore, be a useful tool for investigating the conformational change that 24

occurs when the immature virus enters an acidic environment. During dengue virus 25

maturation, the spiky, immature, non-infectious virions change their structure to smooth-26

surfaced particles in the slightly acid environment of the trans-Golgi network, thereby 27

allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions 28

become fully infectious when they release the cleaved precursor peptide on reaching the 29

neutral pH environment of the extracellular space. Here we report on the cryo-electron 30

microscopy structures of the immature virus complexed with the 2H2 antigen binding 31

fragments (Fab) at different concentrations and varied pH conditions. At neutral pH and 32

high concentration of the Fab molecules, three Fab molecules bind to three precursor-33

membrane proteins on each spike of the immature virus. However, at a low concentration 34

of the Fab molecules and at pH 7.0, only two Fab molecules bind to each spike. Changing to 35

slightly acidic pH caused no detectable change of structure for the high Fab concentration 36

sample, but caused severe structural damage to the low concentration sample. Therefore, 37

the 2H2 Fab inhibits the maturation process of immature dengue virus when the Fab 38

molecules are at high concentration, because the three Fab molecules on each spike hold 39

the precursor-membrane molecules together, thereby inhibiting the normal conformational 40

change that occurs during maturation. 41

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INTRODUCTION 44

Dengue virus (DENV) is a lipid-enveloped, positive-stranded RNA virus that is a member of 45

Flaviviridae. Mosquitos are the major vector for DENV transmission to humans. Symptoms of 46

primary infection are febrile and non-fatal, whereas secondary infections lead to life threatening 47

symptoms such as hemorrhagic fever or dengue shock syndrome (4). The severity of the 48

secondary DENV infection may be associated with antibody-dependent enhancement of 49

infection (ADE) (3, 7). 50

DENV is assembled initially on the endoplasmic reticulum of cells in an immature non-51

infectious form. The fully infectious mature virus is not formed until it is released from its host 52

(24, 25). Both immature (26) and mature (11) DENV particles have icosahedral symmetry with 53

diameters of about 600Å and 500Å with spiky and smooth surfaces, respectively. The structural 54

proteins of DENV are the capsid protein, the precursor-membrane (prM) protein and the 55

envelope (E) protein. The latter two are membrane anchored and are involved in structural re-56

arrangements during maturation and fusion. The prM molecule is a chaperone protein that helps 57

E to fold and to form a heterodimer with prM. The 180 copies of prM-E heterodimers then 58

assemble into 60 trimeric spikes of the immature virus (26). In this form, the pr peptide is located 59

on the top of each trimeric spike, burying much of the fusion loop underneath it (12). Immature 60

DENV are further processed in the trans-Golgi network where the acidic environment causes the 61

immature particles to change into mature-like particles. This structural re-arrangement makes the 62

furin (a cellular protease) cleavage site on prM accessible, resulting in the cleavage of prM, 63

leaving the M protein anchored in the membrane and the pr peptide protecting the fusion loop. 64

The pr peptide is released when the virus is secreted into the neutral pH extracellular space (24, 65


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25). However, DENV maturation is often incomplete with both immature and partially mature 66

virus particles then released into the extracellular space (9, 15). 67

The 2H2 mouse monoclonal antibody was developed (8) and characterized (8, 17) as a 68

highly cross-reactive, low neutralizing antibody that binds to immature DENV. Many other 69

immature DENV antibodies are isolated from patient serum and are agents that can cause ADE 70

(2, 5, 16). Here we report the cryo-electron microscopy (cryoEM) structures of 2H2 Fab 71

fragments complexed with immature virus at different concentrations and pH. We also report the 72

crystal structure of the 2H2 Fab fragment and use it to interpret the structures of the virus-Fab 73

complexes. 74

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MATERIALS AND METHODS 76

Immature DENV preparation. C6/36 mosquito cells were grown in modified Eagle medium 77

(MEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino 78

acids and 20mM HEPES at 28ºC. Infection of DENV-2 (strain P16681) was carried out when the 79

cells were 50-60% confluent at a MOI of 0.5. Culture medium with 20mM NH4Cl was applied to 80

cells 20 hours post infection to stop the virus maturation process. Supernatant was collected, 81

clarified 48 hours post infection and precipitated with 8% PEG overnight. The PEG precipitant 82

was collected, re-suspended in NTE buffer (NaCl 120mM, Tris 20mM EDTA 1mM, pH 8.0) and 83

further purified by a potassium tartrate step gradient centrifugation. The visible virus band at 20 84

to 25% potassium tartrate concentration was extracted and the buffer was exchanged with NTE 85

buffer five times using a Millipore Centricon (100 KDa MW cut-off). The final volume of 86

immature DENV preparation was 100 to 150 μl. The quality of the virus was evaluated by 87


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inspection of the structural protein bands on a SDS PAGE gel. The concentration of the virus was 88

estimated by comparison of the protein band intensities with BSA standards. 89

2H2 Fab production, crystallization and structure determination. Hybridoma cells 90

expressing the 2H2 antibody were obtained from the American Tissue Culture Collection 91

(ATCC) and grown in BD Cell Mab Basal Medium (BD Biosciences) supplemented with 25% 92

fetal bovine serum. The BD CELLine 1000 culture system (BD Biosciences) was used for 93

antibody production. Antibody containing medium was collected every 7 days for 3 weeks. 94

The 2H2 antibody was first purified with a protein A affinity column. Later, the Fab 95

fragment was generated by papain digestion at 37˚C for 6 hrs and separated from the Fc fragment 96

by using a protein A affinity column. The 2H2 Fab was finally purified with a Superdex 75 97

(16/60) column. 98

Purified 2H2 Fab at 10mg/ml was used to set up crystallization screens using the Emerald 99

Wizard I to IV kits (Emerald Biosystems). Crystals were further optimized using the hanging 100

drop method with 25% PEG 6000, 0.1M MES pH 6.0, 0.2M NH4Cl. X-ray diffraction data were 101

collected at the APS beamline 23ID-D using a wavelength of 1.03Å. The diffraction data was 102

indexed and scaled using the HKL2000 program (14). The crystals had a space group of P22121 103

with cell dimensions a=51.55 Å, b= 87.71 Å, c=85.31 Å and diffracted to 1.8Å resolution. The 104

variable and constant domains derived from an HIV Fab (PDB accessing number: 3OZ9) were 105

used as search models to determine the structure by molecular replacement using the program 106

MOLREP in the CCP4 suite of programs (20-22). This HIV antibody was used for the search 107

procedure because it was also a mouse antibody that belonged to the IgG2a family. The 2H2 108

antibody amino acid sequences of the light and heavy chain variable domains were determined 109

by Syd Labs, Inc. The structure was refined (Table 1) using the Phoenix program (1). The final 110


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Rworking and Rfree values are 17.9% and 23.4% using the data to 2.3 Å resolution. A total of 155 111

water molecules were included. 112

Virus Fab complex formation. The 2H2 Fab was added to ~100µl of immature virus at 113

pH 7.0 in phosphate buffer at 4˚C. High (~50 x) and low (~1x) ratios of the Fab molecule relative 114

to each prM molecule in the virus were used for incubating the virus-Fab complex for several 115

hours. The approximate relative ratios of these concentrations were estimated from the intensity 116

of stain on a SDS PAGE gel (Fig. 1). Aliquots of these samples were flash-frozen on Quantifoil 117

holey carbon grids by hand blotting in a biosafety cabinet. The pH of these samples was then 118

lowered in steps of 0.2 to pH 6.0 and aliquots were again used to prepare cryoEM grids. Finally 119

the pH was returned to pH 7.0 in one step and used to prepare cryoEM grids. Each pH change 120

was performed by centrifuging the sample using a Millipore Centricon (100 KDa MW cut-off) at 121

10,000 rpm for 10 min at 4˚C. 122

Data collection and single particle reconstruction. Images of each sample were taken 123

on a CM200 FEG transmission electron microscope (Philips/FEI) at a magnification of 51,000 124

under low-dose conditions (~20 e/Å2) and recorded on Kodak SO-163 films. The micrographs 125

were digitized using a Nikon 9000 scanner with 6.35μm step size. Particles were selected 126

manually with the boxer program in EMAN (13, 19). The microscope contrast transfer function 127

parameters for each micrograph were first determined using an automated fitting method (23) 128

and then manually verified and corrected using the EMAN ctfit graphic program. To avoid model 129

bias, 5 “random” initial models per dataset were generated by random particle orientations 130

assignment. Iterative refinement processes including 2-D particle alignments and 3-D 131

icosahedral reconstructions were performed using the program jspr.py (6) with the 132

EMAN/EMAN2 program (13, 19). The Fourier shell correlation (FSC) between structures built 133


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from the five independent models was then calculated to evaluate the convergence. Among the 134

converged datasets, particles with stable orientations and centers were kept for further 135

refinement. 136

For the immature DENV complexed with high concentration Fabs at pH 7.0, pH 6.0 and 137

back neutralized to pH 7.0, 676, 694 and 680 particles were used for the initial image 138

reconstructions, respectively. The final reconstructions utilized 378 (Fig. 2a to c; Fig. 3a and b), 139

152 (Fig.2g to i), 344 (map not shown) particles, respectively. The resolutions of these maps 140

were estimated to be 21Å, 25Å and 21Å (respectively) based on the resolution at which the FSC 141

became less than 0.5. For the immature DENV complexed with a low Fab concentration at pH 142

7.0, two datasets with 2518 and 4635 particle images were used for the initial reconstruction. The 143

final reconstruction used 802 (map not shown) and 2253 (Fig. 2d to f; Fig. 3c and d) of these 144

particles that produced maps whose resolution was estimated to be 23Å and 21Å, respectively. 145

Structure analysis. The 12.5Å resolution, cryoEM map of the immature DENV (EMDB 146

accessing number: 5422) had been interpreted using the prM-E heterodimer crystal structure 147

(PDB accession number: 3C6D) in terms of 60 trimeric spikes per viron. The structure of the 148

prM-E trimer (12) was used as a single rigid body to fit into the cryoEM map of the various 149

complexes using the EMfit program (18). The density of these cryoEM maps was then set to zero 150

at all grid points that were within 3Å of any atom in the fitted structure. The resultant maps were 151

used to fit the crystal structure of the 2H2 Fab fragments into each of the three independent 152

positions on the glycoprotein spike within the icosahedral asymmetric unit using the EMfit 153

program. The three positions were identified as green, blue and magenta, with the blue position 154

being between the green and magenta positions. The surface areas between neighboring Fab 155

molecules was calculated by the program PISA (10). 156


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Protein structure and cryo-EM density map accession numbers. The structure factors 157

and atomic coordinates of 2H2 Fab were deposited with the Protein Data Bank, accesssion num-158

ber 4KVC. The cryo-EM density maps of the 2H2 Fab complexed with immature dengue virus at 159

different pH conditions and Fab concentrations were deposited with the EM Data Bank, acces-160

sion numbers EMD-5674, EMD-5675, EMD-5676, and EMD-5677. The fitted atomic coordi-161

nates complex has been deposited with the Protein Data Bank, accession number XXXX. 162

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RESULTS 164

The 2H2 Fab fragments were complexed with immature virus at neutral pH for cryoEM data 165

collection. Complexes were formed both when the Fab was at high and at low concentration. 166

These complexes were then moved to a low pH environment for cryoEM studies. The complexes 167

were back neutralized for the final cryoEM studies. However, the low concentration Fab virus 168

complexes disintegrated when the pH was lowered and could therefore be studied only at the 169

initial neutral pH. 170

The cryoEM reconstruction showed that one 2H2 Fab molecule bound to each prM 171

molecule per trimeric spike of the immature virus when a high concentration of Fab molecules 172

was used at neutral pH. In this structure, three Fab densities (green, blue, and magenta) were 173

clearly resolved and were oriented radially outward on each of the trimeric prM-E spikes of the 174

virus (Fig. 2a to c). Thus, a total of 180 copies of 2H2 Fab were bound to the 180 copies of pr on 175

the viral surface. Each of the Fab densities consisted of two lobes corresponding to the constant 176

and variable domain dimers, which were connected by two thin stalks or “elbows” (Fig. 3a and 177

b). The cryoEM density of the virus, underneath the bound Fabs, maintained the features of 178


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immature DENV and this was further confirmed by fitting the prM-E trimer crystal structure 179

(Table 2) into the cryoEM map. 180

The structure of virus Fab complexes with three 2H2 Fabs present on each spike did not 181

change either when the pH was lowered to 6 (Fig. 2g to i) or when subsequently changed back to 182

neutral. Previous results had shown that, in the absence of Fab, lowering the pH from 7.0 to 6.0 183

changed the structure from having 60 spiky trimers to a smooth surfaced virus containing 90 184

dimers. In contrast, it is shown here that the presence of three 2H2 Fab molecules bound to each 185

trimeric spike stopped the conformational change that would have occurred in the absence of 186

bound Fab molecules. 187

However, in the structure of the immature DENV virus complexed with low 188

concentration of Fab at neutral pH, only two Fab densities (green and blue positions) were 189

resolved on each of the trimeric prM-E spikes (Fig. 2d to f and Fig. 3c and d). Thus, 2H2 Fab 190

molecules preferentially bound to two of the three prM molecules per trimeric spike on the viral 191

surface resulting in a total of 120 copies of 2H2 Fab molecules presented. Furthermore, when pH 192

was lowered, these complexes disintegrated into heterogonous particle populations. Therefore 193

the presence of only two Fab molecules on a spike is insufficient to stop the conformational 194

change that normally occurs when immature virus encounters an acidic pH. 195

The crystal structure of the 2H2 Fab fragment was fitted into the cryoEM difference map 196

(Materials and Methods). The binding site of the Fab molecule was on the top of the pr peptide 197

and consisted primarily of the highly exposed a and c strands that belong to two adjacent β-198

sheets (12) of the pr peptide (Fig. 4a and b; Table 3). Both hydrophobic and charged 199

interactions participate in these interactions. Assuming the crystal structure of the isolated prM-E 200

heterodimer (12) to interpret the cryoEM results reported here would place this glycan within 6Å 201


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of the Fab binding interface. However the heterodimer was produced in drosophila cells whereas 202

the virus was propagated in mosquito cells. Thus it is not clear whether the glycan moiety at this 203

site is involved in the binding of the 2H2 antibody. Superposition of the three independent pr 204

peptides in the icosahedral asymmetric unit showed that the Fab fragments bound to the pr 205

peptides are in roughly similar orientation with respect to each prM-E heterodimer (Fig. 4a and 206

b). 207

Even though there are slight differences in the positions and orientations of the three 208

independent Fab molecules relative to each prM-E heterodimer (Fig. 4a and b), there are more 209

significant differences in the Fab occupancies. The central blue Fab is the best ordered as 210

measured by the high density at the atomic positions (sumf) and the low percentage of residues 211

in negative density (-den). In contrast, the magenta Fab is the least ordered and almost 212

completely missing at low Fab concentration (Table 4). The contact area between the blue and 213

green Fab molecules was 525Å2 whereas the contact area between the magenta and blue Fab 214

molecules was only 32Å2 (Fig. 5). Thus the interaction between blue and green Fab molecules 215

may have stabilized their binding to the prM-E molecules even when the Fab concentration was 216

low. 217

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DISCUSSION 219

When the 2H2 Fab molecules were at high concentration, the immature virus did not change its 220

conformation in acid pH (Fig. 2 and Fig. 3), possibly because the association of the three Fab 221

molecules on each prM-E inhibits the conformational change that is required to form the mature 222

virus. In contrast, at low Fab concentration only two Fab molecules (blue and green positions) 223

were bound to each spike. Presumably the association of these two Fab molecules was unable to 224


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hold the spikes together, when the pH was lowered. As a result, the particles degenerated into a 225

heterogeneous collection of conformations that could not be used for a successful image 226

reconstruction. Possibly, at low concentrations, the prM-E heterodimers lacking a Fab molecule 227

would be free to make the first movement on being exposed to acidic pH, but then would not be 228

able to find another unbound partner to make a dimer as required for the formation of a mature 229

particle. These attempts at re-assortment of trimers into dimers would create a large variety of 230

heterogeneous particles as observed. The difference between the low and high concentration 231

structures of the 2H2 Fab complex with partially mature virus may be related to the increase of 232

infectivity caused by low concentration prM binding antibodies (2, 5, 16) and, hence, would also 233

be associated with the occurrence of ADE. Based on the present results, low concentration of 234

2H2 can bind to partially immature virus that would permit the virus to infect other cells by 235

means of their Fc receptor molecules, thus enhancing the infectivity of the virus. 236

Although the spikes in immature flaviviruses are “trimers”, they do not have an exact 3-237

fold axis, showing that the three prM-E hetrodimers in a spike could have slightly different 238

structures and properties. This asymmetry has been amplified in that the Fab bound to the blue 239

site has greater order at all pH and all Fab concentrations (Table 4). The lack of equivalence 240

between the three heterodimers in a spike must originate in the assembly process, suggesting a 241

sequential pathway for the assembly of the immature virus. This lack of equivalence between 242

heterodimers might also be required in the subsequent conformational processes ending up in a 243

mature structure in which the three heterodimers do not have equivalent T=3 environments. 244

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ACKNOWLEDGEMENTS 246

This work was supported by NIH grant AI76331 to MGR. Use of the Advanced Photon Source 247

was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy 248

Sciences, under Contract No. DE-AC02-06CH11357. We thank the National Institutes of Health 249

(S10RR023011A) and Purdue University for their support of the EM facility. 250

We appreciate TJ Battisti’s support in training on cryoEM data collection procedure. We 251

are grateful to Sheryl Kelly for her administrative support. We thank Richard Kuhn for helpful 252

discussions, and Valorie Bowman and Agustin Avila-Sakar for technical support. 253

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FIGURE LEGENDS 343

FIG. 1. SDS-PAGE gel showing the concentration ratio of 2H2 Fab to prM. (a) Immature DENV 344

complexed with high concentration of 2H2 Fab. (b) Immature DENV complexed with low 345

concentration of 2H2 Fab. 346

347

FIG 2. Cryo EM density maps of immature DENV complexed with 2H2 Fab fragments. (a, b, c) 348

High Fab concentration at pH 7.0; (d, e, f) Low Fab concentration at pH 7.0; (g, h, i) High Fab 349

concentration at pH 6.0. (a, d, g) Density map of the whole particle. (b, e, h) Enlarged view 350

showing one asymmetric unit identified by the white triangle. (c, f, i) Side view showing the Fab 351

fragment bound to a trimeric prM-E spike. The maps are colored by radius as indicated. Note 352

that one of the three Fab fragments is missing in d, e and f (see inside the dashed circle) at low 353

concentration of the Fab molecules. 354

355

FIG 3. Structure of the 2H2 Fab crystal structures shown as ribbon drawings fitted into cryoEM 356

density (colored by radius in mesh) of the virus-Fab complex. (a, b) high concentration of the 357

Fab molecules at pH 7.0. (c, d) Low concentration of the Fab molecules at pH 7.0. The three 358

independent positions are colored green, blue and magenta. (a, c) Top view showing one 359

asymmetric unit. (b, d) Side view showing one trimeric spike of the virus-Fab complex. The 360

prM-E trimer is colored baby blue. 361

362

FIG 4. Superposition in pairs of the three 2H2 Fab molecule complexed with the pr peptide of 363

the immature virus, aligned by superimposing the pr peptide. (a) Superposition of the green and 364

blue Fab molecules. (b) Superposition of the blue and magenta Fab molecules. The pr peptide is 365


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shown in baby blue. Note that all 2H2 Fab molecules are in close proximity to the β-sheet 366

structure of the pr peptide. 367

368

FIG 5. Contacts between bound Fab molecules fitted into the map of the immature dengue virus 369

at pH 7.0 complexed with the Fab molecules at high concentration. (a) Side view of the green 370

and blue Fab molecules. (b) Side view of the blue and magenta Fab molecules. The pr peptide is 371

shown in baby blue. 372

373


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TABLES 374

TABLE 1. 2H2 Fab data collection and refinement statistics. 375

Data Collection

Beamline APS 23ID-D

Temperature 100K

Wavelength(Å) 1.03

Resolution(Å) a 2.3

Space group P22121

Unit cell(Å) a = 51.55 b = 87.71 c = 85.31

Unique reflections 16,607

Redundancy 9.5

I/σ 24.7 (3.6)

Completeness(%) 99.9 (100.0)

Rmerge(%)b 10.5

Refinement

Resolution range(Å) 44.1-2.3

Rwork(%)c 17.90

Rfree(%)d 23.35

Average B factor (Å2) 24.42

Rmsd bonds from idealized values (Å) 0.008

Rmsd angels from idealized values (◦) 1.20

Residues in disallowed region of the Ramachandran plot (%)

0.7

a Values in parentheses throughout the table correspond to the outermost resolution shell

b Rmerge =∑ | I- <I> | / ∑ I, where I is measured intensity for reflections with indices hkl. c Rwork= ∑ ||Fobs| - |Fcalc|| ⁄ ∑ |Fobs|

d Rfree has the same formula as Rwork except that calculation was made with the structure factors from the test set.

376


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TABLE 2. Fitting of the prM-E trimer into cryoEM density maps. 377

Fab concentration pH sumfa clashb -denc θ1d θ2 θ3 cxe cy cz

0 8.0 45.3 4.5 1.3 329.5 -0.7 300.0 46.9 50.2 212.6

high 7.0 50.9 0.2 2.2 331.0 1.0 300.0 43.7 50.2 208.4

high 6.0 38.1 0.2 2.5 331.0 1.0 300.0 43.9 49.8 208.6

high bk7.0f 50.2 0.1 3.2 331.0 1.0 300.0 44.0 50.4 208.6

low 7.0 41.3 0.2 1.7 331.0 0.0 300.0 44.3 50.5 209.6asumf is the average density for all atom positions normalized by the highest density in the map 378

to 100. 379

bclash (%) describes the percentage of atoms in the map that have steric clashes with symmetry-380

related subunits. 381

c-den (%) represents the percentage of atoms that are positioned outside of the density. 382

dθ1, θ2, and θ3 (°) are the Eulerian angles that rotate the molecules from their initial position to 383

their fitted positions. 384

ecx, cy and cz (Å) are the final center positions of the molecules after fitting. 385

fbk7.0 represents back neutralized to pH 7.0. 386

387


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TABLE 3. Amino acids in the interface between the virus and Fab molecule. 388

pr 2H2 Fab Heavy Chain

1F Y102

3L Y102

21K S30, S31

24L F32, Y102

25F Y102, P103,

26K N101, Y102, P103, H104, Y49

27T Y102, P103

28E H104

389

390


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TABLE 4. Fitting of the 2H2 Fab structure into the cryoEM density maps. 391

Fab

position

Fab

concentration pH sumfa -denb θ1

c θ2 θ3 cxd cy cz

blue high 7.0 34.0 4.2 121.0 49.5 289.8 72.1 66.9 284.0

high 6.0 24.7 6.9 129.8 52.0 290.0 71.7 66.3 284.5

high bk7.0e 33.1 2.8 121.0 51.0 297.0 72.0 67.0 294.1

low 7.0 18.1 4.6 121.0 50.0 299.0 71.6 67.1 284.0

green high 7.0 32.6 4.1 129.0 47.8 221.3 33.5 91.2 284.2

high 6.0 23.5 8.5 139.3 49.8 201.0 29.3 90.3 284.0

high bk7.0 30.8 5.1 135.3 49.0 218.0 33.6 90.9 282.7

low 7.0 17.1 7.2 141.0 40.0 210.3 33.7 91.7 283.3

magenta high 7.0 27.4 9.8 150.0 63.0 92.0 47.2 28.4 295.1

high 6.0 20.3 17.9 150.3 70.0 90.0 47.2 27.9 296.1

high bk7.0 28.1 8.5 149.8 63.0 92.0 47.3 27.8 295.8

low 7.0 8.6 34.8 159.0 62.0 98.5 47.4 28.4 295.8asumf is the average density for all atom positions normalized by the highest density in the map 392

to 100. 393

b-den (%) represents the percentage of atoms that are positioned outside of the density. 394

cθ1, θ2, and θ3 (°) are the Eulerian angles that rotate the molecules from their initial position to 395

their fitted positions. 396

dcx, cy and cz (Å) are the final center positions of the molecules after fitting. 397

ebk7.0 represents back neutralized to pH 7.0. 398

399

400


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obstruction of dengue virus maturation by fab fragments...

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