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- PDB-7asi: Fixed-target serial femtosecond crystallography using in cellulo ... -

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Basic information

Entry
Database: PDB / ID: 7asi
TitleFixed-target serial femtosecond crystallography using in cellulo grown Neurospora crassa HEX-1 microcrystals. (Chips 1+2)
ComponentseIF-5a domain-containing protein
KeywordsSTRUCTURAL PROTEIN / in cellulo crystals / Woronin body / septal pore sealing
Function / homology
Function and homology information


positive regulation of translational termination / positive regulation of translational elongation / translation elongation factor activity / ribosome binding / RNA binding
Similarity search - Function
Hex1, S1 domain / Translation elongation factor IF5A-like / Translation elongation factor, IF5A C-terminal / Eukaryotic elongation factor 5A hypusine, DNA-binding OB fold / Translation protein SH3-like domain superfamily / Ribosomal protein L2, domain 2 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
eIF-5a domain-containing protein
Similarity search - Component
Biological speciesNeurospora crassa (fungus)
MethodX-RAY DIFFRACTION / FREE ELECTRON LASER / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.704 Å
Model detailsChip 1+2
AuthorsLahey-Rudolph, J.M. / Schoenherr, R. / Barthelmess, M. / Fischer, P. / Seuring, C. / Wagner, A. / Meents, A. / Redecke, L.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research05K18FLA Germany
German Research Foundation (DFG)EXC306 Germany
Joachim Herz StiftungPhD scholarship Germany
CitationJournal: Iucrj / Year: 2021
Title: Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals.
Authors: Lahey-Rudolph, J.M. / Schonherr, R. / Barthelmess, M. / Fischer, P. / Seuring, C. / Wagner, A. / Meents, A. / Redecke, L.
History
DepositionOct 27, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 23, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 31, 2024Group: Advisory / Data collection ...Advisory / 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 / pdbx_unobs_or_zero_occ_atoms
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: eIF-5a domain-containing protein


Theoretical massNumber of molelcules
Total (without water)19,2221
Polymers19,2221
Non-polymers00
Water1,69394
1
A: eIF-5a domain-containing protein

A: eIF-5a domain-containing protein


Theoretical massNumber of molelcules
Total (without water)38,4432
Polymers38,4432
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation11_655-x+y+1,y,-z+1/21
Buried area1930 Å2
ΔGint-7 kcal/mol
Surface area15210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.730, 58.730, 192.830
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Components on special symmetry positions
IDModelComponents
11A-274-

HOH

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Components

#1: Protein eIF-5a domain-containing protein


Mass: 19221.742 Da / Num. of mol.: 1 / Mutation: Met1_Tyr2insGly; Ser175_*insAla
Source method: isolated from a genetically manipulated source
Details: Recombinant baculovirus production in Escherichia coli DH10EmBacY (Geneva Biotech, BacToBac system). Lipofection with Escort IV reagent. P3 recombinant baculovirus stock used for infection ...Details: Recombinant baculovirus production in Escherichia coli DH10EmBacY (Geneva Biotech, BacToBac system). Lipofection with Escort IV reagent. P3 recombinant baculovirus stock used for infection of insect cells at MOI 1.
Source: (gene. exp.) Neurospora crassa (fungus) / Gene: GE21DRAFT_1527
Plasmid: modified pFastBac1 vector containing the sequence 5 -ATGGGCGCCTAA-3 between the BamHI and HindIII restriction sites
Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0B0EDT5
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 94 / Source method: isolated from a natural source / Formula: H2O
Compound detailsThe in cellulo crystallized Neurospora crassa HEX-1 forms a polymeric helical spiral with 12 ...The in cellulo crystallized Neurospora crassa HEX-1 forms a polymeric helical spiral with 12 monomers per full turn, or 6 homodimeric units per full turn. The homodimeric assembly features an identical interaction on opposite faces that propagates the spiral. This results in the six-fold symmetry of the crystals that is consistently observed in native Woronin bodies

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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: 2.51 Å3/Da / Density % sol: 50.93 %
Crystal growTemperature: 300.15 K / Method: in cell
Details: Neurospora crassa HEX-1 P3 rBV-infected Sf9 cells at multiplicity of infection 1, cultivated in serum-free ESF921 medium

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Data collection

DiffractionMean temperature: 298 K / Serial crystal experiment: Y
Diffraction sourceSource: FREE ELECTRON LASER / Site: SLAC LCLS / Beamline: MFX / Wavelength: 1.312 Å
DetectorType: CS-PAD CXI-1 / Detector: PIXEL / Date: Oct 2, 2017 / Details: Beryllium lenses
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.312 Å / Relative weight: 1
ReflectionResolution: 1.704→39.9 Å / Num. obs: 22635 / % possible obs: 99.99 % / Redundancy: 124.12 % / Biso Wilson estimate: 18.31 Å2 / CC1/2: 0.964 / CC star: 0.991 / R split: 0.162 / Net I/σ(I): 5.421
Reflection shellResolution: 1.704→1.744 Å / Redundancy: 16.4 % / Mean I/σ(I) obs: 0.7 / Num. unique obs: 1469 / CC1/2: 0.16 / CC star: 0.525 / R split: 0.189 / % possible all: 99.86
Serial crystallography measurementCollection time total: 0.25 hours / Focal spot size: 1 µm2 / Pulse duration: 27.5 fsec. / Pulse photon energy: 9.45 keV / Source distance: 0.116 m / XFEL pulse repetition rate: 120 Hz
Serial crystallography sample deliveryDescription: fixed target / Method: fixed target
Serial crystallography sample delivery fixed targetDescription: micro-patterned single-crystalline silicon microchips
Details: scanning speed matched to 120 Hz pulse repetition rate
Motion control: attachment to Roadrunner II / Sample dehydration prevention: humidor, helium chamber / Sample solvent: ESF921 insect cell medium / Sample unit size: 12 µm / Support base: Chips glued to aluminium support frame / Velocity horizontal: 2.5 / Velocity vertical: 100
Serial crystallography data reductionCrystal hits: 19676 / Frames failed index: 6286 / Frames indexed: 13390 / Frames total: 87450 / Lattices indexed: 15224 / XFEL pulse events: 87450 / XFEL run numbers: 6,9

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIXv1.16-3546refinement
PHENIXv1.9-1692refinement
PHASERv1.16-3546phasing
PDB_EXTRACT3.25data extraction
CrystFELv0.6.3data processing
CrystFELv0.6.3data scaling
Cheetahdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1khi

1khi


Resolution: 1.704→29.365 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 22.95 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2643 1787 7.95 %
Rwork0.2189 20683 -
obs0.2225 22470 99.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 87.52 Å2 / Biso mean: 25.3897 Å2 / Biso min: 3.79 Å2
Refinement stepCycle: final / Resolution: 1.704→29.365 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1093 0 0 94 1187
Biso mean---35.11 -
Num. residues----141
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0121137
X-RAY DIFFRACTIONf_angle_d1.3161546
X-RAY DIFFRACTIONf_chiral_restr0.058182
X-RAY DIFFRACTIONf_plane_restr0.006202
X-RAY DIFFRACTIONf_dihedral_angle_d14.684427
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.704-1.75010.50261310.4541152098
1.7501-1.80160.33441340.35531542100
1.8016-1.85970.34251340.27681542100
1.8597-1.92620.26571340.23761553100
1.9262-2.00330.26991340.2051560100
2.0033-2.09440.24161360.1771581100
2.0944-2.20480.2491360.17451552100
2.2048-2.34290.21111370.1641590100
2.3429-2.52370.22821360.17681576100
2.5237-2.77750.2271380.20021605100
2.7775-3.1790.27251400.21011615100
3.179-4.00360.28721430.22791655100

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