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- PDB-6rum: Crystal structure of GFP-LAMA-G97 - a GFP enhancer nanobody with ... -

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Basic information

Entry
Database: PDB / ID: 6rum
TitleCrystal structure of GFP-LAMA-G97 - a GFP enhancer nanobody with cpDHFR insertion and TMP and NADPH
ComponentsGFP-LAMA-G97 a GFP enhancer nanobody with cpDHFR insertion
KeywordsPROTEIN BINDING / LAMA / nanobody / circular permutant of DHFR / TMP / NADPH
Function / homology
Function and homology information


methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process ...methotrexate binding / dihydrofolic acid binding / response to methotrexate / NADP+ binding / folic acid binding / dihydrofolate metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / NADPH binding / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / response to xenobiotic stimulus / response to antibiotic / cytosol
Similarity search - Function
Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase-like domain superfamily
Similarity search - Domain/homology
Chem-NDP / DI(HYDROXYETHYL)ETHER / TRIMETHOPRIM / Dihydrofolate reductase
Similarity search - Component
Biological specieslama glama (llama)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsFarrants, H. / Tarnawski, M. / Mueller, T.G. / Otsuka, S. / Hiblot, J. / Koch, B. / Kueblbeck, M. / Kraeusslich, H.-G. / Ellenberg, J. / Johnsson, K.
CitationJournal: Nat.Methods / Year: 2020
Title: Chemogenetic Control of Nanobodies.
Authors: Farrants, H. / Tarnawski, M. / Muller, T.G. / Otsuka, S. / Hiblot, J. / Koch, B. / Kueblbeck, M. / Krausslich, H.G. / Ellenberg, J. / Johnsson, K.
History
DepositionMay 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jan 24, 2024Group: 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

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Assembly

Deposited unit
A: GFP-LAMA-G97 a GFP enhancer nanobody with cpDHFR insertion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,4278
Polymers30,9851
Non-polymers1,4427
Water5,314295
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3020 Å2
ΔGint-30 kcal/mol
Surface area13080 Å2
MethodPISA
Unit cell
Length a, b, c (Å)47.890, 56.580, 53.290
Angle α, β, γ (deg.)90.00, 94.91, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Antibody , 1 types, 1 molecules A

#1: Antibody GFP-LAMA-G97 a GFP enhancer nanobody with cpDHFR insertion


Mass: 30984.646 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) lama glama (llama), (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: folA, tmrA, b0048, JW0047 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABQ4, dihydrofolate reductase

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Non-polymers , 6 types, 302 molecules

#2: Chemical ChemComp-TOP / TRIMETHOPRIM


Mass: 290.318 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H18N4O3 / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
#3: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#6: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 295 / 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: 2.32 Å3/Da / Density % sol: 47.02 %
Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 0.1 M MES pH 6.0, 20% (w/v) PEG 6000, 1.0 M lithium chloride

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 0.99999 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 19, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.99999 Å / Relative weight: 1
ReflectionResolution: 1.6→50 Å / Num. obs: 37239 / % possible obs: 99.1 % / Redundancy: 3.3 % / Rmerge(I) obs: 0.054 / Net I/σ(I): 13.23
Reflection shellResolution: 1.6→1.7 Å / Redundancy: 3.2 % / Rmerge(I) obs: 0.49 / Mean I/σ(I) obs: 2.43 / Num. unique obs: 6123 / % possible all: 98.6

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Processing

Software
NameVersionClassification
PHENIX(1.14rc1_3177: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5UII, 5H8D

5uii

5h8d


Resolution: 1.6→47.715 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 24.47
RfactorNum. reflection% reflection
Rfree0.2275 1862 5 %
Rwork0.183 --
obs0.1852 37239 99.11 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.6→47.715 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2167 0 92 295 2554
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0062338
X-RAY DIFFRACTIONf_angle_d0.9573183
X-RAY DIFFRACTIONf_dihedral_angle_d5.4151864
X-RAY DIFFRACTIONf_chiral_restr0.061329
X-RAY DIFFRACTIONf_plane_restr0.006411
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.64330.3251410.26412682X-RAY DIFFRACTION98
1.6433-1.69160.2721420.23782694X-RAY DIFFRACTION99
1.6916-1.74620.27761410.23122691X-RAY DIFFRACTION99
1.7462-1.80860.30631450.21822753X-RAY DIFFRACTION99
1.8086-1.88110.26861420.20322701X-RAY DIFFRACTION99
1.8811-1.96670.20721430.17822716X-RAY DIFFRACTION100
1.9667-2.07040.21011430.17042719X-RAY DIFFRACTION99
2.0704-2.20010.22261440.17392724X-RAY DIFFRACTION100
2.2001-2.36990.20671430.18212716X-RAY DIFFRACTION99
2.3699-2.60840.24911430.18922726X-RAY DIFFRACTION99
2.6084-2.98580.26931450.19452760X-RAY DIFFRACTION99
2.9858-3.76160.22271440.16732724X-RAY DIFFRACTION99
3.7616-47.73580.18361460.16482771X-RAY DIFFRACTION98

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