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- PDB-3n9g: Crystal structure of the Fab fragment of the human neutralizing a... -

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Basic information

Entry
Database: PDB / ID: 3n9g
TitleCrystal structure of the Fab fragment of the human neutralizing anti-West Nile Virus MAb CR4354
Components
  • Fab fragment of MAb CR4354, heavy chain
  • Fab fragment of MAb CR4354, light chain
KeywordsIMMUNE SYSTEM / FAB fragment / human neutralizing antibody / MAb CR4354 / anti-west nile virus
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Function and homology information
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.434 Å
AuthorsKaufmann, B. / Rossmann, M.G.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2010
Title: Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354.
Authors: Bärbel Kaufmann / Matthew R Vogt / Jaap Goudsmit / Heather A Holdaway / Anastasia A Aksyuk / Paul R Chipman / Richard J Kuhn / Michael S Diamond / Michael G Rossmann /
Abstract: Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a ...Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.
History
DepositionMay 29, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 24, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 8, 2017Group: Advisory / Refinement description / Category: pdbx_unobs_or_zero_occ_atoms / software
Revision 1.3Dec 6, 2017Group: Database references / Category: pdbx_database_related
Item: _pdbx_database_related.content_type / _pdbx_database_related.db_id
Revision 2.0Dec 25, 2019Group: Derived calculations / Polymer sequence
Category: entity_poly / pdbx_struct_mod_residue / struct_conn
Item: _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_struct_mod_residue.parent_comp_id / _struct_conn.pdbx_leaving_atom_flag
Revision 2.1Mar 31, 2021Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.pdbx_host_org_cell_line / _entity_src_gen.pdbx_host_org_strain
Revision 2.2Sep 6, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Fab fragment of MAb CR4354, heavy chain
L: Fab fragment of MAb CR4354, light chain


Theoretical massNumber of molelcules
Total (without water)47,6542
Polymers47,6542
Non-polymers00
Water14,790821
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3360 Å2
ΔGint-24 kcal/mol
Surface area20270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.741, 71.068, 118.597
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Antibody Fab fragment of MAb CR4354, heavy chain


Mass: 24745.699 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: transient co-expression with entity 2 / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293T / Production host: Homo sapiens (human)
#2: Antibody Fab fragment of MAb CR4354, light chain


Mass: 22908.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: transient co-expression with entity 1 / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293T / Production host: Homo sapiens (human)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 821 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.42 Å3/Da / Density % sol: 49.18 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6
Details: reservoir solution: 20% PEG8000, 10mM MES buffer, pH6.0, 200mM Ca(OAc)2, starting protein concentration in drop: 8 mg/ml, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.0332 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Apr 24, 2010
Details: 300x300mm2 active area, ~2.2sec read-time, 4096x4096 of 0.072mm pixels
RadiationMonochromator: Double crystal cryo-cooled Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 1.43→50 Å / Num. all: 78075 / Num. obs: 78075 / % possible obs: 90.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.7 % / Rmerge(I) obs: 0.072 / Χ2: 2.22 / Net I/σ(I): 13.6
Reflection shellResolution: 1.43→1.45 Å / Redundancy: 2.7 % / Rmerge(I) obs: 0.542 / Mean I/σ(I) obs: 1.63 / Num. unique all: 1674 / Χ2: 1.041 / % possible all: 39.4

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 57.53 / Model details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å45.53 Å
Translation2.5 Å45.53 Å

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASER2.1.4phasing
PHENIX1.5_2refinement
PDB_EXTRACT3.1data extraction
HKL-2000data collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: WAM homology model of variable domain + constant domain of PDB ID 3KYK

3kyk


Resolution: 1.434→45.531 Å / Occupancy max: 1 / Occupancy min: 0 / FOM work R set: 0.887 / SU ML: 0.17 / σ(F): 1.34 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.186 3868 4.96 %RANDOM
Rwork0.167 ---
obs0.168 77999 91.3 %-
all-78002 --
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 38.821 Å2 / ksol: 0.339 e/Å3
Displacement parametersBiso max: 93.53 Å2 / Biso mean: 19.731 Å2 / Biso min: 4.39 Å2
Baniso -1Baniso -2Baniso -3
1-1.441 Å20 Å2-0 Å2
2--0.771 Å2-0 Å2
3----2.211 Å2
Refinement stepCycle: LAST / Resolution: 1.434→45.531 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3349 0 0 821 4170
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0063623
X-RAY DIFFRACTIONf_angle_d1.1284984
X-RAY DIFFRACTIONf_chiral_restr0.073566
X-RAY DIFFRACTIONf_plane_restr0.005644
X-RAY DIFFRACTIONf_dihedral_angle_d16.3841331
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 28

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.434-1.4520.285570.2671236129343
1.452-1.470.268980.2491552165054
1.47-1.490.2541030.2411700180361
1.49-1.510.272930.2331926201967
1.51-1.5310.231160.2132089220573
1.531-1.5540.2031170.2032305242280
1.554-1.5790.1961230.1912459258286
1.579-1.6050.2411410.1762678281993
1.605-1.6320.2031390.1712839297898
1.632-1.6620.1981590.16528583017100
1.662-1.6940.1841620.15828683030100
1.694-1.7280.181560.15328513007100
1.728-1.7660.2031440.15828803024100
1.766-1.8070.2121460.16128573003100
1.807-1.8520.1911290.15629073036100
1.852-1.9020.181670.15528783045100
1.902-1.9580.171460.15428893035100
1.958-2.0220.1791430.15428883031100
2.022-2.0940.1651270.15629403067100
2.094-2.1780.1941660.15628963062100
2.178-2.2770.1741480.15528903038100
2.277-2.3970.1751560.16429013057100
2.397-2.5470.1831590.1729133072100
2.547-2.7430.1911630.17129203083100
2.743-3.020.1691510.16329363087100
3.02-3.4560.1611490.15429833132100
3.456-4.3540.151470.14129853132100
4.354-45.5540.1831630.1683107327099
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.7382-0.3237-0.36441.2629-0.09660.86130.01580.05390.0103-0.1836-0.05310.03360.0273-0.07960.0310.11430.0377-0.02410.0915-0.01520.0685-15.8512-11.448510.1125
21.1203-0.10160.04831.19290.79371.06190.0460.1537-0.0720.00720.0749-0.05820.08680.1052-0.09670.09450.0298-0.01090.0874-0.03150.06751.4038-15.933445.3879
30.8348-0.15880.31780.3418-0.18491.7370.0115-0.0030.0342-0.0487-0.04150.048-0.0612-0.06350.02540.05790.0106-0.01940.03360.00470.0792-28.95841.146822.6355
40.26650.21190.13930.67780.12840.57250.01850.00150.0331-0.0742-0.0229-0.00660.01050.07230.00380.03720.0014-0.00310.0649-0.00140.0481-4.948-3.687942.8727
50.09290.0267-0.34250.0849-0.06461.73530.12450.19220.1530.14940.07360.0744-0.5940.4971-0.16220.2065-0.01690.06180.15440.00740.178313.32764.562744.9457
60.49540.3673-0.23081.38680.59581.0210.0234-0.15220.17660.1728-0.01810.07-0.07080.1031-0.00750.0933-0.01850.00140.09010.00210.1019-2.28294.804247.4865
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain L and resid 1:113)L1 - 113
2X-RAY DIFFRACTION2(chain L and resid 114:220)L114 - 220
3X-RAY DIFFRACTION3(chain H and resid 1:123)H1 - 123
4X-RAY DIFFRACTION4(chain H and resid 124:195)H124 - 195
5X-RAY DIFFRACTION5(chain H and resid 196:199)H196 - 199
6X-RAY DIFFRACTION6(chain H and resid 200:230)H200 - 230

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