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- PDB-5u0u: Crystal Structure of DH270.1 (unliganded, single-chain Fv) from t... -

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Basic information

Entry
Database: PDB / ID: 5u0u
TitleCrystal Structure of DH270.1 (unliganded, single-chain Fv) from the DH270 Broadly Neutralizing N332-glycan Dependent Lineage
ComponentsDH270.1 single-chain variable fragment
KeywordsIMMUNE SYSTEM / single-chain variable fragment / HIV-1 / ANTIBODY
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Function and homology information
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.428 Å
AuthorsFera, D. / Harrison, S.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1F32AI116355-01 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM-1- AI100645 United States
CitationJournal: Sci Transl Med / Year: 2017
Title: Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies.
Authors: Mattia Bonsignori / Edward F Kreider / Daniela Fera / R Ryan Meyerhoff / Todd Bradley / Kevin Wiehe / S Munir Alam / Baptiste Aussedat / William E Walkowicz / Kwan-Ki Hwang / Kevin O ...Authors: Mattia Bonsignori / Edward F Kreider / Daniela Fera / R Ryan Meyerhoff / Todd Bradley / Kevin Wiehe / S Munir Alam / Baptiste Aussedat / William E Walkowicz / Kwan-Ki Hwang / Kevin O Saunders / Ruijun Zhang / Morgan A Gladden / Anthony Monroe / Amit Kumar / Shi-Mao Xia / Melissa Cooper / Mark K Louder / Krisha McKee / Robert T Bailer / Brendan W Pier / Claudia A Jette / Garnett Kelsoe / Wilton B Williams / Lynn Morris / John Kappes / Kshitij Wagh / Gift Kamanga / Myron S Cohen / Peter T Hraber / David C Montefiori / Ashley Trama / Hua-Xin Liao / Thomas B Kepler / M Anthony Moody / Feng Gao / Samuel J Danishefsky / John R Mascola / George M Shaw / Beatrice H Hahn / Stephen C Harrison / Bette T Korber / Barton F Haynes /
Abstract: A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and ...A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.
History
DepositionNov 27, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 15, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2017Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Refinement description / Category: pdbx_audit_support / software
Item: _pdbx_audit_support.funding_organization / _software.name
Revision 1.3Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Oct 4, 2023Group: 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: DH270.1 single-chain variable fragment
A: DH270.1 single-chain variable fragment
C: DH270.1 single-chain variable fragment
E: DH270.1 single-chain variable fragment


Theoretical massNumber of molelcules
Total (without water)109,2044
Polymers109,2044
Non-polymers00
Water0
1
G: DH270.1 single-chain variable fragment


Theoretical massNumber of molelcules
Total (without water)27,3011
Polymers27,3011
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: DH270.1 single-chain variable fragment


Theoretical massNumber of molelcules
Total (without water)27,3011
Polymers27,3011
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: DH270.1 single-chain variable fragment


Theoretical massNumber of molelcules
Total (without water)27,3011
Polymers27,3011
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
E: DH270.1 single-chain variable fragment


Theoretical massNumber of molelcules
Total (without water)27,3011
Polymers27,3011
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)156.873, 111.284, 89.134
Angle α, β, γ (deg.)90.000, 120.460, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain C
31chain E
41chain G

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: GLN / Beg label comp-ID: GLN / End auth comp-ID: HIS / End label comp-ID: HIS / Auth seq-ID: 1 - 249 / Label seq-ID: 1 - 251

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain AAB
2chain CCC
3chain EED
4chain GGA

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Components

#1: Antibody
DH270.1 single-chain variable fragment


Mass: 27300.980 Da / Num. of mol.: 4 / Fragment: FAB
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pVRC-8400 / Cell line (production host): HEK 293T / Production host: Homo sapiens (human)

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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: 3.07 Å3/Da / Density % sol: 59.94 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.5
Details: 2.5 M sodium chloride, 100 mM sodium acetate, pH 4.5 and 200 mM LiSO4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97925 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Oct 27, 2016
RadiationMonochromator: SINGLE CRYSTAL SI(220) SIDE BOUNCE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97925 Å / Relative weight: 1
ReflectionResolution: 3.42→49.851 Å / Num. obs: 14420 / % possible obs: 81.5 % / Redundancy: 2.3 % / Biso Wilson estimate: 60.08 Å2 / Rmerge(I) obs: 0.184 / Rpim(I) all: 0.14 / Rrim(I) all: 0.233 / Χ2: 0.986 / Net I/av σ(I): 4.603 / Net I/σ(I): 3.8 / Num. measured all: 32526
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsCC1/2Diffraction-ID% possible all
3.42-3.482.20.6520.503185.5
3.48-3.542.20.4860.603183.6
3.54-3.612.20.4340.688184
3.61-3.682.20.3940.621181.6
3.68-3.762.20.3450.778184.3
3.76-3.852.30.3320.802182.3
3.85-3.952.20.2980.814183.1
3.95-4.052.30.2970.821180.6
4.05-4.172.30.2280.888182
4.17-4.312.20.1920.92182.7
4.31-4.462.30.1690.926181.4
4.46-4.642.30.1740.929181.2
4.64-4.852.30.1510.935181.9
4.85-5.112.30.150.942181.1
5.11-5.432.30.1420.946181.6
5.43-5.852.30.1450.943179.7
5.85-6.432.30.1440.947179.8
6.43-7.362.30.1460.94179.2
7.36-9.262.30.1040.962179.8
9.26-502.40.0890.971175.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation6.89 Å49.85 Å
Translation6.89 Å49.85 Å

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
PHASER2.5.7phasing
PARROTphasing
PHENIX1.8.2_1309refinement
Cootmodel building
PDB_EXTRACT3.2data extraction
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5TQA

5tqa


Resolution: 3.428→49.851 Å / SU ML: 0.45 / Cross valid method: FREE R-VALUE / σ(F): 1.4 / Phase error: 27.43
RfactorNum. reflection% reflection
Rfree0.282 726 5.04 %
Rwork0.2536 --
obs0.255 14409 80.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 103.1 Å2 / Biso mean: 61.17 Å2 / Biso min: 39.23 Å2
Refinement stepCycle: final / Resolution: 3.428→49.851 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7157 0 0 0 7157
Num. residues----940
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0047353
X-RAY DIFFRACTIONf_angle_d0.89210001
X-RAY DIFFRACTIONf_chiral_restr0.0361072
X-RAY DIFFRACTIONf_plane_restr0.0051280
X-RAY DIFFRACTIONf_dihedral_angle_d12.8072553
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4210X-RAY DIFFRACTION10.777TORSIONAL
12C4210X-RAY DIFFRACTION10.777TORSIONAL
13E4210X-RAY DIFFRACTION10.777TORSIONAL
14G4210X-RAY DIFFRACTION10.777TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 5

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.4283-3.69290.33441280.32042700282880
3.6929-4.06440.34081590.29232771293083
4.0644-4.65220.26131320.24882774290682
4.6522-5.85980.2471430.22812752289581
5.8598-49.8560.2621640.21782686285079
Refinement TLS params.Method: refined / Origin x: 117.0391 Å / Origin y: -44.3423 Å / Origin z: 125.2583 Å
111213212223313233
T0.3581 Å2-0.0217 Å2-0.0106 Å2-0.3815 Å2-0.044 Å2--0.4435 Å2
L-0.0714 °20.0304 °2-0.1013 °2-0.8812 °2-1.606 °2--2.5421 °2
S-0.0836 Å °-0.0207 Å °-0.0537 Å °-0.136 Å °0.0268 Å °-0.0579 Å °0.2488 Å °-0.0498 Å °0.0724 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allG1 - 249
2X-RAY DIFFRACTION1allA1 - 249
3X-RAY DIFFRACTION1allC1 - 249
4X-RAY DIFFRACTION1allE1 - 249

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