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- PDB-7sak: Crystal Structure of LaM4 Nanobody bound to mCherry -

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Basic information

Entry
Database: PDB / ID: 7sak
TitleCrystal Structure of LaM4 Nanobody bound to mCherry
Components
  • LaM4
  • mCherry
KeywordsIMMUNE SYSTEM / Antibody / nanobody / affinity tag / fluorescent protein
Function / homologyDI(HYDROXYETHYL)ETHER
Function and homology information
Biological speciesDiscosoma sp. (sea anemone)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.15 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: mCherry
B: LaM4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,6494
Polymers42,5082
Non-polymers1422
Water8,431468
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2290 Å2
ΔGint-17 kcal/mol
Surface area14930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)156.554, 41.920, 47.827
Angle α, β, γ (deg.)90.000, 94.370, 90.000
Int Tables number5
Space group name H-MC121
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Components on special symmetry positions
IDModelComponents
11B-252-

HOH

21B-291-

HOH

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Components

#1: Protein mCherry


Mass: 26996.393 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Discosoma sp. (sea anemone) / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2
#2: Antibody LaM4


Mass: 15511.113 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
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 468 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.84 Å3/Da / Density % sol: 33.21 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 4
Details: 20 % PEG3350, 100 mM MMT (Malic Acid, MES and TRIS)

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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: Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 1.15→75 Å / Num. obs: 207109 / % possible obs: 97 % / Redundancy: 9.6 % / Rmerge(I) obs: 0.082 / Rpim(I) all: 0.027 / Rrim(I) all: 0.086 / Χ2: 1.016 / Net I/σ(I): 11.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.15-1.195.90.41784750.910.1810.4560.87677.3
1.19-1.247.90.355102410.9480.1320.3790.92793.9
1.24-1.39.90.294109040.9730.0970.311.02499.4
1.3-1.3610.20.239108870.9780.0770.2511.02699.6
1.36-1.459.60.186109990.9850.0630.1961.05999.9
1.45-1.5610.20.14109380.9920.0460.1481.044100
1.56-1.7210.50.109110120.9940.0350.1141.033100
1.72-1.979.80.085109790.9950.0290.091.038100
1.97-2.4810.40.072110820.9960.0230.0761.017100
2.48-7510.30.061112580.9980.020.0641.00999.9

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Processing

Software
NameVersionClassification
PHENIX1.17refinement
DENZO720data reduction
SCALEPACK720data scaling
PHASER1.17phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2H5Q

2h5q


Resolution: 1.15→42.15 Å / SU ML: 0.08 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 13.11 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1439 10288 4.97 %
Rwork0.1173 196620 -
obs0.1186 206908 95.31 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 65.6 Å2 / Biso mean: 16.0711 Å2 / Biso min: 2.6 Å2
Refinement stepCycle: final / Resolution: 1.15→42.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2725 0 15 481 3221
Biso mean--20.9 32.94 -
Num. residues----345
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.15-1.160.2391900.21063583377352
1.16-1.170.19372660.19025044531073
1.17-1.190.23162480.17335587583581
1.19-1.20.16953450.16125936628187
1.2-1.220.15513020.14356457675993
1.22-1.240.14923410.1376533687495
1.24-1.250.15653460.13686696704298
1.25-1.270.18163610.1366771713297
1.27-1.290.16673220.13396783710599
1.29-1.310.17383270.12646690701798
1.31-1.340.16644040.12096778718299
1.34-1.360.14323650.11446829719498
1.36-1.390.15613420.10696844718699
1.39-1.410.13173470.1046758710599
1.41-1.450.14183850.10456817720299
1.45-1.480.13873640.100167907154100
1.48-1.520.13393470.09986816716399
1.52-1.560.12753510.10196852720399
1.56-1.60.12933590.09886791715099
1.6-1.650.14163540.10776855720999
1.65-1.710.1483670.10676801716899
1.71-1.780.13663670.10246831719899
1.78-1.860.12833670.09976854722199
1.86-1.960.13263430.09996781712499
1.96-2.080.11963620.102569027264100
2.08-2.250.12923440.107568637207100
2.25-2.470.12963810.112968197200100
2.47-2.830.14413580.118768577215100
2.83-3.560.14433650.122168527217100
3.56-42.150.15463680.133668507218100

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