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

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Basic information

Entry
Database: PDB / ID: 7asx
TitleFixed-target serial femtosecond crystallography using in cellulo grown Neurospora crassa HEX-1 microcrystals. (Chip 1)
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 C-terminal / Eukaryotic elongation factor 5A hypusine, DNA-binding OB fold / Translation elongation factor IF5A-like / 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: 1.8 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 28, 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
Water2,306128
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 area1860 Å2
ΔGint-10 kcal/mol
Surface area15020 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.750, 58.750, 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-223-

HOH

21A-278-

HOH

31A-302-

HOH

41A-326-

HOH

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Components

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


Mass: 19221.742 Da / Num. of mol.: 1 / Mutation: Ser175_*insAla
Source method: isolated from a genetically manipulated source
Details: baculovirus production in Escherichia coli DH10EmBacY cells (Geneva Biotech). 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


Mass: 18.015 Da / Num. of mol.: 128 / 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: 2.5 Å3/Da / Density % sol: 50.78 %
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: 1.799→39.9 Å / Num. obs: 19213 / % possible obs: 99.95 % / Redundancy: 107.66 % / Biso Wilson estimate: 17.22 Å2 / CC1/2: 0.951 / CC star: 0.9873 / R split: 0.1722 / Net I/σ(I): 5.17
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. unique obsCC1/2CC starR split% possible all
1.799-1.84927.61.314470.3650.73120.95100
1.849-1.936.41.8414280.59250.86260.6353100
1.903-1.96546.92.4314550.73950.92210.4514100
1.9646-2.034672.13.4614330.86060.96180.3059100
2.0345-2.115993.34.2614440.89210.97110.2389100
2.1159-2.21241164.9914470.93020.98180.1941100
2.2124-2.329110.75.4614660.92090.97920.1773100
2.329-2.47535.6714640.91430.97740.1815100
2.4753-2.666114.614770.9160.97780.1666100
2.666-2.93415010.91490.97750.1534
2.934-3.359127.67.3314920.90510.97480.1447100
3.359-4.23146.98.9915560.91560.97770.1223100
Serial crystallography measurementCollection time total: 0.12 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: Chip glued to aluminium support frame / Velocity horizontal: 2.5 / Velocity vertical: 100
Serial crystallography data reductionCrystal hits: 13247 / Frames indexed: 9726 / Frames total: 43840 / Lattices indexed: 11279 / XFEL run numbers: 9

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Phasing

PhasingMethod: molecular replacement

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Processing

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

1khi


Resolution: 1.8→30.734 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 22.25 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2731 1541 8.02 %
Rwork0.2261 17671 -
obs0.2298 19212 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 99.36 Å2 / Biso mean: 23.9591 Å2 / Biso min: 0.81 Å2
Refinement stepCycle: final / Resolution: 1.8→30.734 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1093 0 0 128 1221
Biso mean---36.48 -
Num. residues----141
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
1.8-1.85810.36671360.36241556
1.8581-1.92450.35071360.28331567
1.9245-2.00160.30481370.23561558
2.0016-2.09260.23171370.20991574
2.0926-2.20290.27271360.21531561
2.2029-2.34090.23581380.18771587
2.3409-2.52160.24061390.18961584
2.5216-2.77520.24561400.20181610
2.7752-3.17640.2621410.21161622
3.1764-4.00050.28571450.23361654

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