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- PDB-6m9i: L-GSTSTA from degenerate octameric repeats in InaZ, residues 707-712 -

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Basic information

Entry
Database: PDB / ID: 6m9i
TitleL-GSTSTA from degenerate octameric repeats in InaZ, residues 707-712
ComponentsIce nucleation proteinIce nucleus
KeywordsPROTEIN FIBRIL / amyloid / racemic / ice nucleation / MicroED / InaZ / pseudomonas syringae
Function / homologyBacterial ice-nucleation, octamer repeat / Ice nucleation protein repeat / Bacterial ice-nucleation proteins octamer repeat. / ice binding / cell outer membrane / Ice nucleation protein
Function and homology information
Biological speciesPseudomonas syringae pv. syringae (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 0.9 Å
AuthorsZee, C. / Glynn, C. / Gallagher-Jones, M. / Miao, J. / Santiago, C.G. / Cascio, D. / Gonen, T. / Sawaya, M.R. / Rodriguez, J.A.
Funding support United States, 2items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103403 United States
CitationJournal: IUCrJ / Year: 2019
Title: Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ.
Authors: Chih-Te Zee / Calina Glynn / Marcus Gallagher-Jones / Jennifer Miao / Carlos G Santiago / Duilio Cascio / Tamir Gonen / Michael R Sawaya / Jose A Rodriguez /
Abstract: The ice-nucleation protein InaZ from contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. structures of this repeat segment, ...The ice-nucleation protein InaZ from contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. structures of this repeat segment, resolved to 1.1 Å by microfocus X-ray crystallography and to 0.9 Å by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals.
History
DepositionAug 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 27, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / diffrn_radiation_wavelength / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_radiation_wavelength.wavelength / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Assembly

Deposited unit
A: Ice nucleation protein


Theoretical massNumber of molelcules
Total (without water)5231
Polymers5231
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area770 Å2
Unit cell
Length a, b, c (Å)9.190, 11.890, 22.430
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein/peptide Ice nucleation protein / Ice nucleus / inaZ


Mass: 522.508 Da / Num. of mol.: 1 / Fragment: UNP residues 707-712 / Source method: obtained synthetically / Details: L-GSTSTA
Source: (synth.) Pseudomonas syringae pv. syringae (bacteria)
References: UniProt: P06620
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: L-GSTSTA from ice nucleation protein inaZ / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Pseudomonas syringae pv. syringae (bacteria)
EM crystal formationDetails: Batch Mode: 0.1 M McIlvaine, pH 4.2, 12.5% w/v PEG8000, 0.1 M sodium chloride
Temperature: 298 K
Buffer solutionpH: 4.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.01 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)
EM diffractionCamera length: 1156 mm
EM diffraction shellResolution: 7.27→0.9 Å / Fourier space coverage: 86.4 % / Multiplicity: 4.3 / Num. of structure factors: 1747 / Phase residual: 1.0E-10 °
EM diffraction statsDetails: Phase statistics are not applicable. No imaging was used. The phases were obtained using a crystallographic direct methods program, SHELXD.
Fourier space coverage: 86.4 % / High resolution: 0.9 Å / Num. of intensities measured: 7494 / Num. of structure factors: 1750 / Phase error: 0 ° / Phase residual: 1.0E-10 ° / Phase error rejection criteria: not applicable / Rmerge: 16.2 / Rsym: 16.2
DetectorDate: Sep 24, 2017
RadiationScattering type: electron
Radiation wavelengthRelative weight: 1
ReflectionResolution: 0.9→7.271 Å / Num. obs: 1760 / % possible obs: 85.5 % / Redundancy: 4.269 % / Biso Wilson estimate: 42.62 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.162 / Rrim(I) all: 0.185 / Χ2: 0.962 / Net I/σ(I): 5.5 / Num. measured all: 7513 / Scaling rejects: 1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
0.9-14.2060.3573.0919015404520.7260.40983.7
1-1.14.4710.2234.4213953543120.9690.25288.1
1.1-1.24.520.2185.349992532210.9440.24687.4
1.2-1.34.5510.2036.167191801580.9580.2387.8
1.3-1.54.2390.1946.39242472180.950.22288.3
1.5-1.84.1650.1597.586831881640.9740.18187.2
1.8-2.33.9750.1058.964771451200.9890.12282.8
2.3-33.7420.1059.0324778660.9860.12284.6
3-53.50.0829.6214053400.9920.09275.5
5-7.2713.1110.1069.03282090.9970.12645

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
PHENIX1.12_2829refinement
PDB_EXTRACT3.24data extraction
XDSdata reduction
SHELXDphasing
EM software
IDNameVersionCategory
6Coot0.8.9.1model fitting
8PHENIX1.12_2829model refinement
12XSCALEcrystallography merging
13SHELXD3D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 9.19 Å / B: 11.89 Å / C: 22.43 Å / Space group name: P212121 / Space group num: 19
CTF correctionType: NONE
3D reconstructionResolution: 0.9 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: maximum likelihood
RefinementResolution: 0.9→0.9 Å / SU ML: 0.05 / Cross valid method: THROUGHOUT / σ(F): 1.45 / Phase error: 20.67
RfactorNum. reflection% reflection
Rfree0.2324 173 9.9 %
Rwork0.2173 --
obs0.2188 1747 86.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 3.94 Å2 / Biso mean: 1.169 Å2 / Biso min: 0.13 Å2
Refinement stepCycle: final / Resolution: 0.902→7.271 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms36 0 0 1 37
Biso mean---3.28 -
Num. residues----6
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00935
ELECTRON CRYSTALLOGRAPHYf_angle_d1.05747
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0377
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0046
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d7.54210
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Rfactor Rfree error: 0 / Total num. of bins used: 8

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
0.9022-0.94320.2866200.304918620682
0.9432-0.99280.374220.292719421690
0.9928-1.05480.2908200.230619021087
1.0548-1.1360.3107220.226219421689
1.136-1.24980.2244220.228519621887
1.2498-1.42950.1737220.196519822088
1.4295-1.79650.2374230.199120622988
1.7965-7.27150.1731220.187721023283

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