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- PDB-7sai: Crystal Structure of Lag30 Nanobody bound to eGFP -

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Basic information

Entry
Database: PDB / ID: 7sai
TitleCrystal Structure of Lag30 Nanobody bound to eGFP
Components
  • Green fluorescent protein
  • LAG30 Nanobody
KeywordsIMMUNE SYSTEM / Antibody / nanobody / affinity tag / fluorescent protein
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / PHOSPHATE ION / Green fluorescent protein
Function and homology information
Biological speciesAequorea victoria (jellyfish)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.23 Å
AuthorsCong, A.T.Q. / Schellenberg, M.J.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: Protein Sci. / Year: 2022
Title: High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.
Authors: Cong, A.T.Q. / Witter, T.L. / Schellenberg, M.J.
History
DepositionSep 22, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.3Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Green fluorescent protein
C: LAG30 Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,88812
Polymers42,8522
Non-polymers1,03610
Water3,333185
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1450 Å2
ΔGint-3 kcal/mol
Surface area15640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)173.172, 173.172, 92.288
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number97
Space group name H-MI422
Components on special symmetry positions
IDModelComponents
11A-304-

PO4

21A-304-

PO4

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Components

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Protein / Antibody , 2 types, 2 molecules AC

#1: Protein Green fluorescent protein


Mass: 26882.299 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Production host: Escherichia coli (E. coli) / Strain (production host): Rodetta2 / References: UniProt: P42212
#2: Antibody LAG30 Nanobody


Mass: 15969.824 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2

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Non-polymers , 4 types, 195 molecules

#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#5: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 185 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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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: 4.11 Å3/Da / Density % sol: 70.05 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: 800 mM KH2PO4/NaH2PO4, 100 mM HEPES

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 29, 2020
RadiationMonochromator: SI111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.2→50 Å / Num. obs: 65199 / % possible obs: 100 % / Redundancy: 13.6 % / Rmerge(I) obs: 0.146 / Rpim(I) all: 0.041 / Rrim(I) all: 0.151 / Χ2: 1.017 / Net I/σ(I): 7.5
Reflection shell

Diffraction-ID: 1 / % possible all: 100

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2
2.2-2.2813.91.79133530.5040.4961.8590.99
2.28-2.3713.61.35833810.6870.381.4110.99
2.37-2.4812.81.03133770.8020.2981.0731.017
2.48-2.6113.20.71433790.8850.2040.7431.023
2.61-2.7714.40.48234100.960.1310.51.051
2.77-2.9914.10.32534030.9820.0890.3371.035
2.99-3.2913.70.17834190.9920.050.1851.034
3.29-3.7612.50.12134400.9950.0360.1261.002
3.76-4.7414.40.09534720.9960.0260.0990.981
4.74-50130.08836240.9970.0250.0921.042

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
DENZOdata reduction
SCALEPACKdata scaling
PHASER1.17.1_3660phasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4EUL

4eul


Resolution: 2.23→48.94 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 22.26 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1971 3274 5.02 %
Rwork0.1711 61918 -
obs0.1724 65192 99.16 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 211.41 Å2 / Biso mean: 65.5829 Å2 / Biso min: 33.2 Å2
Refinement stepCycle: final / Resolution: 2.23→48.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2781 0 62 185 3028
Biso mean--120.87 64.93 -
Num. residues----353
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 23

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.23-2.260.29671240.31662177230181
2.26-2.290.35261330.310527262859100
2.29-2.330.35761430.304427082851100
2.33-2.370.32331510.280127312882100
2.37-2.420.27631370.266527002837100
2.42-2.460.28271460.25526972843100
2.46-2.510.27531400.254527402880100
2.51-2.570.28411520.244327142866100
2.57-2.630.28151400.233127042844100
2.63-2.690.26391400.220727232863100
2.69-2.760.21581470.206427012848100
2.76-2.850.22561430.201627512894100
2.85-2.940.2281420.193626762818100
2.94-3.040.2281380.192627302868100
3.04-3.160.23411520.180127022854100
3.16-3.310.21451450.176527092854100
3.31-3.480.1931430.16327132856100
3.48-3.70.17431390.145427372876100
3.7-3.990.17131450.148127052850100
3.99-4.390.13341420.120927052847100
4.39-5.020.15031500.113727282878100
5.02-6.330.16921440.143327062850100
6.33-48.940.18471380.177827352873100

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