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- PDB-4xt1: Structure of a nanobody-bound viral GPCR bound to human chemokine... -

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Basic information

Entry
Database: PDB / ID: 4xt1
TitleStructure of a nanobody-bound viral GPCR bound to human chemokine CX3CL1
Components
  • FractalkineCX3CL1
  • G-protein coupled receptor homolog US28
  • nanobody 7Single-domain antibody
KeywordsVIRAL PROTEIN/SIGNALLING PROTEIN / GPCR / chemokine / membrane protein / complex / VIRAL PROTEIN-SIGNALLING PROTEIN complex
Function / homology
Function and homology information


positive regulation by virus of host cell division / : / CXCR1 chemokine receptor binding / positive regulation of calcium-independent cell-cell adhesion / negative regulation of glutamate receptor signaling pathway / negative regulation of interleukin-1 alpha production / leukocyte adhesive activation / CX3C chemokine receptor binding / negative regulation of hippocampal neuron apoptotic process / autocrine signaling ...positive regulation by virus of host cell division / : / CXCR1 chemokine receptor binding / positive regulation of calcium-independent cell-cell adhesion / negative regulation of glutamate receptor signaling pathway / negative regulation of interleukin-1 alpha production / leukocyte adhesive activation / CX3C chemokine receptor binding / negative regulation of hippocampal neuron apoptotic process / autocrine signaling / synapse pruning / negative regulation of neuron migration / positive regulation of microglial cell migration / negative regulation of microglial cell activation / positive regulation of transforming growth factor beta1 production / regulation of lipopolysaccharide-mediated signaling pathway / chemokine receptor activity / positive regulation of I-kappaB phosphorylation / microglial cell proliferation / positive regulation of actin filament bundle assembly / CCR chemokine receptor binding / leukocyte migration involved in inflammatory response / lymphocyte chemotaxis / integrin activation / eosinophil chemotaxis / angiogenesis involved in wound healing / chemokine-mediated signaling pathway / leukocyte chemotaxis / Chemokine receptors bind chemokines / chemokine activity / negative regulation of cell-substrate adhesion / positive regulation of cell-matrix adhesion / negative regulation of interleukin-1 beta production / neuron remodeling / positive regulation of neuroblast proliferation / positive chemotaxis / chemoattractant activity / monocyte chemotaxis / negative regulation of interleukin-6 production / negative regulation of apoptotic signaling pathway / negative regulation of tumor necrosis factor production / regulation of neurogenesis / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / cellular response to interleukin-1 / extrinsic apoptotic signaling pathway in absence of ligand / virus-mediated perturbation of host defense response / cell chemotaxis / neutrophil chemotaxis / negative regulation of cell migration / positive regulation of release of sequestered calcium ion into cytosol / cell projection / response to ischemia / microglial cell activation / positive regulation of smooth muscle cell proliferation / regulation of synaptic plasticity / defense response / positive regulation of neuron projection development / cell-cell adhesion / positive regulation of inflammatory response / cellular response to type II interferon / neuron cellular homeostasis / cytokine-mediated signaling pathway / chemotaxis / integrin binding / cell-cell signaling / cellular response to tumor necrosis factor / G alpha (i) signalling events / positive regulation of canonical NF-kappaB signal transduction / positive regulation of MAPK cascade / positive regulation of ERK1 and ERK2 cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cell adhesion / neuron projection / immune response / inflammatory response / G protein-coupled receptor signaling pathway / signaling receptor binding / neuronal cell body / positive regulation of cell population proliferation / negative regulation of apoptotic process / host cell plasma membrane / perinuclear region of cytoplasm / cell surface / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular region / membrane / plasma membrane
Similarity search - Function
CX3C chemokine domain / Chemokine receptor family / Chemokine beta/gamma/delta / Intercrine alpha family (small cytokine C-X-C) (chemokine CXC). / Chemokine interleukin-8-like domain / Chemokine interleukin-8-like superfamily / Small cytokines (intecrine/chemokine), interleukin-8 like / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #40 / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins ...CX3C chemokine domain / Chemokine receptor family / Chemokine beta/gamma/delta / Intercrine alpha family (small cytokine C-X-C) (chemokine CXC). / Chemokine interleukin-8-like domain / Chemokine interleukin-8-like superfamily / Small cytokines (intecrine/chemokine), interleukin-8 like / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #40 / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Immunoglobulins / Up-down Bundle / Immunoglobulin-like / Beta Barrel / Sandwich / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
CHOLESTEROL / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / SUCCINIC ACID / Unknown ligand / G-protein coupled receptor homolog US28 / Fractalkine
Similarity search - Component
Biological speciesCytomegalovirus
Homo sapiens (human)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.886 Å
AuthorsBurg, J.S. / Jude, K.M. / Waghray, D. / Garcia, K.C.
CitationJournal: Science / Year: 2015
Title: Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.
Authors: Burg, J.S. / Ingram, J.R. / Venkatakrishnan, A.J. / Jude, K.M. / Dukkipati, A. / Feinberg, E.N. / Angelini, A. / Waghray, D. / Dror, R.O. / Ploegh, H.L. / Garcia, K.C.
History
DepositionJan 22, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 4, 2015Provider: repository / Type: Initial release
Revision 1.1Mar 18, 2015Group: Database references
Revision 2.0Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Polymer sequence / Refinement description / Source and taxonomy
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / entity_poly / entity_src_gen / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _database_2.pdbx_DOI ..._citation.journal_id_CSD / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_alt_source_flag / _pdbx_struct_oper_list.symmetry_operation

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: G-protein coupled receptor homolog US28
B: Fractalkine
C: nanobody 7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,66619
Polymers66,6363
Non-polymers3,03116
Water41423
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7910 Å2
ΔGint-40 kcal/mol
Surface area23920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.024, 81.024, 231.303
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number79
Space group name H-MI4

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein G-protein coupled receptor homolog US28 / HHRF3


Mass: 42041.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cytomegalovirus / Cell line (production host): HEK293s GnTI- / Production host: Homo sapiens (human) / References: UniProt: P69332
#2: Protein Fractalkine / CX3CL1 / C-X3-C motif chemokine 1 / CX3C membrane-anchored chemokine / Neurotactin / Small-inducible cytokine D1


Mass: 9970.462 Da / Num. of mol.: 1 / Fragment: UNP residues 25-101 / Mutation: N9A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CX3CL1, FKN, NTT, SCYD1, A-152E5.2 / Cell line (production host): HEK293s GnTI- / Production host: Homo sapiens (human) / References: UniProt: P78423

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Antibody , 1 types, 1 molecules C

#3: Antibody nanobody 7 / Single-domain antibody


Mass: 14624.158 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21

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Non-polymers , 5 types, 39 molecules

#4: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#5: Chemical
ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C21H40O4
#6: Chemical
ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 7 / Source method: obtained synthetically
#7: Chemical ChemComp-SIN / SUCCINIC ACID / Succinic acid


Mass: 118.088 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H6O4
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 23 / 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: 2

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Sample preparation

CrystalDensity Matthews: 2.85 Å3/Da / Density % sol: 56.8 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 6.3
Details: PEG 300, MES, sodium succinate, polypropylene glycol 400

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.033 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Oct 13, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionResolution: 2.88→50 Å / Num. obs: 16648 / % possible obs: 98.7 % / Redundancy: 8.5 % / Biso Wilson estimate: 58.12 Å2 / Rmerge(I) obs: 0.2 / Rpim(I) all: 0.084 / Rrim(I) all: 0.214 / Χ2: 1.137 / Net I/av σ(I): 10.745 / Net I/σ(I): 5.2 / Num. measured all: 141947
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Num. unique allCC1/2Rpim(I) allΧ2% possible allRmerge(I) obsRrim(I) all
2.88-2.936.86290.3040.5240.87474.8
2.93-2.9888290.4090.4880.79299.8
2.98-3.048.28310.5770.4350.87100
3.04-3.18.48430.6020.4070.89100
3.1-3.178.58400.6760.3320.9371000.8720.936
3.17-3.248.68700.7930.2810.9751000.7460.799
3.24-3.328.68250.7880.2650.9961000.70.75
3.32-3.418.78360.8760.2061.0411000.5450.584
3.41-3.518.88450.8670.1691.0911000.4530.484
3.51-3.638.78310.9170.1391.1311000.3690.395
3.63-3.768.78450.9460.1151.2911000.310.331
3.76-3.918.78540.9670.0911.3121000.2450.262
3.91-4.098.78360.9730.081.3741000.2150.229
4.09-4.38.78510.9790.0651.3841000.1750.187
4.3-4.578.88320.9870.0481.3551000.1310.14
4.57-4.928.78570.9890.0481.3061000.1280.137
4.92-5.428.88430.980.0531.3281000.140.15
5.42-6.28.78390.9820.0581.2781000.1560.166
6.2-7.818.78450.9910.0441.1251000.1230.13
7.81-508.58670.9950.0411.18899.50.1140.122

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Processing

Software
NameVersionClassification
HKL-2000data reduction
PHENIXdev_1839refinement
PDB_EXTRACT3.15data extraction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4MBS, 1F2L, 3ONA, 4B41

4mbs

1f2l

3ona

4b41


Resolution: 2.886→38.234 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 27.33 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2492 1659 9.98 %
Rwork0.1991 14970 -
obs0.2043 16629 99.57 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 206.21 Å2 / Biso mean: 66.9984 Å2 / Biso min: 21.81 Å2
Refinement stepCycle: final / Resolution: 2.886→38.234 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3700 0 255 23 3978
Biso mean--78.78 40.45 -
Num. residues----475
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0024044
X-RAY DIFFRACTIONf_angle_d0.6055461
X-RAY DIFFRACTIONf_chiral_restr0.021630
X-RAY DIFFRACTIONf_plane_restr0.003649
X-RAY DIFFRACTIONf_dihedral_angle_d12.8011462
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 12

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.8856-2.97050.33541250.27231181130695
2.9705-3.06630.32191370.256212371374100
3.0663-3.17590.28381370.234112531390100
3.1759-3.30290.33731390.230712451384100
3.3029-3.45320.29841400.233812641404100
3.4532-3.63510.30411450.20212331378100
3.6351-3.86270.24721420.190412601402100
3.8627-4.16060.2281370.199912511388100
4.1606-4.57860.21791350.157312611396100
4.5786-5.23970.20221340.169912651399100
5.2397-6.5960.25341450.221212471392100
6.596-38.23740.20831430.184612731416100
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)
13.61810.2164-0.48897.7641-4.01434.42860.2541-1.0191-0.15010.5337-0.2781-0.09370.7752-0.27180.05520.7263-0.15520.10930.7106-0.0240.44897.83417.9238277.5957
29.5434-5.94222.66837.01061.51954.06990.8410.39120.44460.2966-0.21470.9728-0.9919-1.3048-1.09790.6665-0.1040.2870.9599-0.01220.434890.16121.1182275.9458
36.69412.005-4.21323.7743-3.49897.16390.0082-0.6647-0.63790.65930.04-0.09010.7415-0.53070.00460.8674-0.15890.06380.6620.06750.298799.579218.4164278.1598
46.05710.09430.52723.28091.04166.4363-0.17040.98670.5568-0.48510.4170.1129-0.7431-0.4589-0.03350.6672-0.05850.10040.67450.01310.5254110.283323.3554207.6492
52.3303-0.6707-1.72441.74640.19463.9634-0.25860.1193-0.4502-0.01470.07220.17270.7907-0.31020.19150.357-0.05850.00520.2609-0.04550.4665103.099110.5731240.6328
62.94390.3258-0.38391.45951.60128.8955-0.22360.22170.0628-0.1167-0.15450.4046-0.0129-0.62110.3990.19230.0114-0.02230.3210.03530.422992.475622.3415242.3529
73.4908-2.867-1.47588.56945.69728.4630.34791.4016-0.1867-1.3734-1.37321.9016-0.4287-0.84831.01440.8177-0.0693-0.1481.0753-0.20030.661292.340720.5767214.98
80.949-0.0388-0.02740.53890.16355.3558-0.07090.0897-0.046-0.10620.03170.0247-0.1538-0.07030.02760.3088-0.05110.00690.27870.0360.394106.31929.7905237.0153
97.7503-1.34823.26675.3358-4.89888.03260.4807-0.1203-0.6189-0.2217-0.5434-0.60090.86590.41130.14230.38430.0627-0.07270.4173-0.07910.6507109.69629.3779257.7165
104.55982.3673-3.10256.5817-1.90829.65180.32710.9673-0.0434-0.6332-0.39161.6379-0.87140.1197-0.10660.59540.0365-0.06280.8527-0.05730.5501102.872521.594212.199
110.4372-0.8465-1.41042.06152.98884.7053-1.04610.9625-0.18611.76390.09760.80730.9873-3.34181.14141.2398-0.117-0.1321.6698-0.08280.7576101.483424.0413188.1388
123.7392-1.8308-3.66851.2740.88755.7859-0.72491.4117-1.20020.58171.1974-0.72362.007-2.3345-0.10251.8237-0.4539-0.14821.6473-0.34010.8598102.399314.3923196.3178
131.94951.8877-1.59292.9397-1.17353.0594-0.45060.2093-0.2632-1.10370.21790.1150.5901-0.1105-0.36041.0706-0.0774-0.13221.9454-0.0630.456100.947820.6617199.6775
149.6854-7.614-1.415.99221.32036.38530.18893.4295-2.502-0.608-0.32632.15311.5941-2.3030.67761.2548-0.7648-0.0632.1225-0.03471.102896.035313.8865190.405
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 66 )A0
2X-RAY DIFFRACTION2chain 'A' and (resid 67 through 72 )A0
3X-RAY DIFFRACTION3chain 'A' and (resid 73 through 116 )A0
4X-RAY DIFFRACTION4chain 'C' and (resid 16 through 33 )C0
5X-RAY DIFFRACTION5chain 'C' and (resid 34 through 103 )C0
6X-RAY DIFFRACTION6chain 'C' and (resid 104 through 169 )C0
7X-RAY DIFFRACTION7chain 'C' and (resid 170 through 183 )C0
8X-RAY DIFFRACTION8chain 'C' and (resid 184 through 290 )C0
9X-RAY DIFFRACTION9chain 'C' and (resid 291 through 310 )C0
10X-RAY DIFFRACTION10chain 'D' and (resid 2 through 16 )D0
11X-RAY DIFFRACTION11chain 'D' and (resid 17 through 23 )D0
12X-RAY DIFFRACTION12chain 'D' and (resid 24 through 29 )D0
13X-RAY DIFFRACTION13chain 'D' and (resid 30 through 57 )D0
14X-RAY DIFFRACTION14chain 'D' and (resid 58 through 69 )D0

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