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- PDB-6rul: Crystal structure of GFP-LAMA-F98 - a GFP enhancer nanobody with ... -

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Basic information

Entry
Database: PDB / ID: 6rul
TitleCrystal structure of GFP-LAMA-F98 - a GFP enhancer nanobody with cpDHFR insertion and TMP and NADPH
ComponentsGFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,GFP-LAMA-F98 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 / 10-formyltetrahydrofolate biosynthetic process / response to methotrexate / folic acid biosynthetic process / folic acid binding / NADP+ binding / dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity ...methotrexate binding / dihydrofolic acid binding / 10-formyltetrahydrofolate biosynthetic process / response to methotrexate / folic acid biosynthetic process / folic acid binding / NADP+ binding / dihydrofolate metabolic 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, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / Immunoglobulins ...Dihydrofolate reductase / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / Immunoglobulins / Immunoglobulin-like / Sandwich / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Chem-NDP / TRIETHYLENE GLYCOL / TRIMETHOPRIM / Dihydrofolate reductase
Similarity search - Component
Biological speciesLama glama (llama)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 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
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,1714
Polymers30,9851
Non-polymers1,1863
Water93752
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2530 Å2
ΔGint1 kcal/mol
Surface area12940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)86.610, 86.610, 73.040
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number171
Space group name H-MP62

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Components

#1: Antibody GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,Dihydrofolate reductase,GFP-LAMA-F98 a GFP enhancer nanobody with cpDHFR insertion,Dihydrofolate reductase,GFP-LAMA-F98 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
#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-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 52 / Source method: isolated from a natural source / Formula: H2O
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: 2.55 Å3/Da / Density % sol: 51.81 %
Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 0.1 M MES pH 6.0, 30% (v/v) PEG 600, 5% (w/v) PEG 1000, 10% (v/v) glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 0.97797 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 10, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97797 Å / Relative weight: 1
ReflectionResolution: 2.2→50 Å / Num. obs: 15911 / % possible obs: 99.9 % / Redundancy: 10.1 % / Rmerge(I) obs: 0.058 / Net I/σ(I): 22.62
Reflection shellResolution: 2.2→2.3 Å / Redundancy: 10.4 % / Rmerge(I) obs: 0.453 / Mean I/σ(I) obs: 5.32 / Num. unique obs: 1970 / % possible all: 99.9

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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: 2.2→43.305 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 24.27
RfactorNum. reflection% reflection
Rfree0.2195 796 5 %
Rwork0.1747 --
obs0.177 15910 99.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.2→43.305 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2167 0 79 52 2298
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0082304
X-RAY DIFFRACTIONf_angle_d1.0683133
X-RAY DIFFRACTIONf_dihedral_angle_d6.1661331
X-RAY DIFFRACTIONf_chiral_restr0.058323
X-RAY DIFFRACTIONf_plane_restr0.006405
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2001-2.33790.30011310.22462497X-RAY DIFFRACTION100
2.3379-2.51840.27481330.20722518X-RAY DIFFRACTION100
2.5184-2.77180.23871310.20242498X-RAY DIFFRACTION100
2.7718-3.17280.2641330.20782514X-RAY DIFFRACTION100
3.1728-3.9970.24661330.16762525X-RAY DIFFRACTION100
3.997-43.31340.16461350.15012562X-RAY DIFFRACTION100

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