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- PDB-5uix: Crystal Structure of the DH576 CD4bs Fab (unliganded) from the RV... -

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Basic information

Entry
Database: PDB / ID: 5uix
TitleCrystal Structure of the DH576 CD4bs Fab (unliganded) from the RV305 HIV Vaccine Trial
Components
  • DH576 Fab heavy chain
  • DH576 Fab light chain
KeywordsIMMUNE SYSTEM / FAB 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: 2.501 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
Bill & Melinda Gates FoundationOPP1033098 United States
CitationJournal: PLoS Pathog / Year: 2017
Title: Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial.
Authors: David Easterhoff / M Anthony Moody / Daniela Fera / Hao Cheng / Margaret Ackerman / Kevin Wiehe / Kevin O Saunders / Justin Pollara / Nathan Vandergrift / Rob Parks / Jerome Kim / Nelson L ...Authors: David Easterhoff / M Anthony Moody / Daniela Fera / Hao Cheng / Margaret Ackerman / Kevin Wiehe / Kevin O Saunders / Justin Pollara / Nathan Vandergrift / Rob Parks / Jerome Kim / Nelson L Michael / Robert J O'Connell / Jean-Louis Excler / Merlin L Robb / Sandhya Vasan / Supachai Rerks-Ngarm / Jaranit Kaewkungwal / Punnee Pitisuttithum / Sorachai Nitayaphan / Faruk Sinangil / James Tartaglia / Sanjay Phogat / Thomas B Kepler / S Munir Alam / Hua-Xin Liao / Guido Ferrari / Michael S Seaman / David C Montefiori / Georgia D Tomaras / Stephen C Harrison / Barton F Haynes /
Abstract: The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is ...The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains.
TRIAL REGISTRATION: ClinicalTrials.gov NCT01435135.
History
DepositionJan 15, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Refinement description / Category: pdbx_audit_support / software
Item: _pdbx_audit_support.funding_organization / _software.name
Revision 1.2Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 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
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: DH576 Fab heavy chain
L: DH576 Fab light chain


Theoretical massNumber of molelcules
Total (without water)48,3192
Polymers48,3192
Non-polymers00
Water1,54986
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3240 Å2
ΔGint-22 kcal/mol
Surface area19660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.638, 149.725, 74.108
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221

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Components

#1: Antibody DH576 Fab heavy chain


Mass: 24942.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Strain: HEK 293T / Cell line (production host): HEK 293T / Production host: Homo sapiens (human)
#2: Antibody DH576 Fab light chain


Mass: 23376.893 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK 293T / Production host: Homo sapiens (human)
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 86 / 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: 2.45 Å3/Da / Density % sol: 49.7 % / Mosaicity: 1.316 °
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 20% PEG 4000, 100mM Hepes, pH 7.0, 1M NaCl

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97925 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Apr 6, 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: 2.51→50 Å / Num. obs: 16590 / % possible obs: 96.6 % / Redundancy: 4 % / Biso Wilson estimate: 32.92 Å2 / Rmerge(I) obs: 0.219 / Rpim(I) all: 0.115 / Rrim(I) all: 0.248 / Χ2: 1.036 / Net I/σ(I): 3.1 / Num. measured all: 65840
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.51-2.552.70.78190.5710.470.8490.5194.9
2.55-2.630.6648010.6240.4070.7850.53195.8
2.6-2.653.60.78280.6470.390.8070.5596.4
2.65-2.73.80.6348140.740.3440.7260.5498.5
2.7-2.7640.5868230.750.3140.6690.56897.5
2.76-2.834.10.5518450.7530.2910.6270.60699.2
2.83-2.94.20.4858240.8020.2510.550.6697.3
2.9-2.984.10.4468220.7870.2390.5090.65997
2.98-3.0640.3838230.8670.2060.4380.70598.7
3.06-3.163.70.3378310.8810.1880.3880.81696.7
3.16-3.284.30.38190.9130.1530.3390.87796.6
3.28-3.414.40.2538240.9380.1270.2851.00496.4
3.41-3.564.30.2338280.9420.1180.2631.15896.6
3.56-3.754.30.2178370.9570.1090.2451.44997.1
3.75-3.984.10.1878320.9620.0970.2121.50297.3
3.98-4.293.90.1518330.9690.0810.1731.64296.9
4.29-4.724.50.1438270.980.070.161.90595.4
4.72-5.414.30.1368460.9760.0680.1541.59295.7
5.41-6.8140.138400.9740.0680.1471.17594.9
6.81-504.10.1048740.990.0540.1181.47293

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation5.62 Å43.82 Å
Translation5.62 Å43.82 Å

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Processing

Software
NameVersionClassification
PHENIX1.8.2_1309refinement
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
PHASER2.5.7phasing
Cootmodel building
PDB_EXTRACT3.22data extraction
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4JM2, 4LSS

4jm2

4lss


Resolution: 2.501→43.819 Å / FOM work R set: 0.8308 / SU ML: 0.23 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 23.9 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2503 815 4.91 %
Rwork0.2111 15773 -
obs0.2131 16588 96.2 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 116.34 Å2 / Biso mean: 53.7 Å2 / Biso min: 20 Å2
Refinement stepCycle: final / Resolution: 2.501→43.819 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3357 0 0 86 3443
Biso mean---49.89 -
Num. residues----439
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0043436
X-RAY DIFFRACTIONf_angle_d0.8884662
X-RAY DIFFRACTIONf_chiral_restr0.061518
X-RAY DIFFRACTIONf_plane_restr0.004598
X-RAY DIFFRACTIONf_dihedral_angle_d13.8951235
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 6

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.5011-2.65770.31791170.28362522263993
2.6577-2.86290.31311410.25832642278398
2.8629-3.15090.30231420.23982616275898
3.1509-3.60670.26361100.21212658276897
3.6067-4.54330.20091510.1812647279897
4.5433-43.82570.2311540.18532688284295
Refinement TLS params.Method: refined / Origin x: -325.8989 Å / Origin y: 168.389 Å / Origin z: 235.8621 Å
111213212223313233
T0.3012 Å2-0.004 Å2-0.0128 Å2-0.2985 Å2-0.0033 Å2--0.3027 Å2
L0.2006 °2-0.0412 °2-0.1087 °2-0.1194 °2-0.078 °2--0.2875 °2
S-0.0012 Å °-0.0089 Å °0.0228 Å °0.0001 Å °-0.0053 Å °-0.028 Å °-0.0194 Å °-0.021 Å °0 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allH1 - 230
2X-RAY DIFFRACTION1allL1 - 213
3X-RAY DIFFRACTION1allS1 - 87

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