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- PDB-8t0b: Novel Domain of Unknown Function Solved with AlphaFold -

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Basic information

Entry
Database: PDB / ID: 8t0b
TitleNovel Domain of Unknown Function Solved with AlphaFold
ComponentsDUF1842 domain-containing protein
KeywordsUNKNOWN FUNCTION / AlphaFold / Bacterial Protein / Domain of Unknown Function / Novel Fold
Function / homologyDomain of unknown function DUF1842 / Domain of unknown function DUF1843 / Domain of unknown function (DUF1842) / Domain of unknown function (DUF1843) / DUF1842 domain-containing protein
Function and homology information
Biological speciesBurkholderia pseudomallei (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsMiller, J.E. / Cascio, D. / Sawaya, M.R. / Yeates, T.O.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2024
Title: AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography.
Authors: Justin E Miller / Matthew P Agdanowski / Joshua L Dolinsky / Michael R Sawaya / Duilio Cascio / Jose A Rodriguez / Todd O Yeates /
Abstract: Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent ...Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.
History
DepositionMay 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._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
Revision 1.2Apr 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DUF1842 domain-containing protein
B: DUF1842 domain-containing protein


Theoretical massNumber of molelcules
Total (without water)46,6292
Polymers46,6292
Non-polymers00
Water19811
1
A: DUF1842 domain-containing protein


Theoretical massNumber of molelcules
Total (without water)23,3141
Polymers23,3141
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: DUF1842 domain-containing protein


Theoretical massNumber of molelcules
Total (without water)23,3141
Polymers23,3141
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)39.470, 40.430, 78.490
Angle α, β, γ (deg.)90.00, 97.01, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein DUF1842 domain-containing protein


Mass: 23314.254 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Burkholderia pseudomallei (bacteria) / Gene: BPSS0212 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q63NT7
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 11 / 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

Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.5 / Details: 25% PEG 3350, 100mM Bis-Tris pH 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 1.4586 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Feb 22, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.4586 Å / Relative weight: 1
ReflectionResolution: 2.1→77.9 Å / Num. obs: 25176 / % possible obs: 89.1 % / Redundancy: 1.62 % / CC1/2: 0.999 / Rmerge(I) obs: 0.042 / Rrim(I) all: 0.059 / Net I/σ(I): 8.85
Reflection shell
Resolution (Å)Rmerge(I) obsNum. unique obsCC1/2Rrim(I) allDiffraction-ID
2.1-2.150.49817120.7660.7011
2.15-2.210.44918450.7350.6311
2.21-2.270.39418150.790.5541
2.27-2.340.32516710.8560.4581
2.34-2.420.28516760.9080.4011
2.42-2.510.25216720.9040.3551
2.51-2.60.19316310.9480.2721
2.6-2.710.16215290.9460.2281
2.71-2.830.10115140.9830.1421
2.83-2.960.08213320.9870.1161
2.96-3.120.06413250.990.091
3.12-3.310.04712550.9930.0671
3.31-3.540.03711270.9960.0521
3.54-3.830.03510260.9970.0491
3.83-4.190.0329400.9960.0451
4.19-4.690.0268690.9970.0371
4.69-5.410.0257740.9980.0351
5.41-6.630.0237000.9980.0321
6.63-9.370.0214840.9990.0291
9.37-77.90.0222790.9990.0311

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XSCALEdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1→77.9 Å / SU ML: 0.35 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 34.47 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2868 1395 10.01 %
Rwork0.2511 --
obs0.2547 13938 95.01 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.1→77.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1752 0 0 11 1763
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0081795
X-RAY DIFFRACTIONf_angle_d0.9482455
X-RAY DIFFRACTIONf_dihedral_angle_d11.572595
X-RAY DIFFRACTIONf_chiral_restr0.064283
X-RAY DIFFRACTIONf_plane_restr0.007314
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.170.34961300.34251167X-RAY DIFFRACTION90
2.17-2.260.38471390.30541248X-RAY DIFFRACTION95
2.26-2.360.33221340.30271222X-RAY DIFFRACTION93
2.36-2.490.34461410.3281262X-RAY DIFFRACTION97
2.49-2.640.36341430.31131284X-RAY DIFFRACTION97
2.64-2.840.37351410.29321269X-RAY DIFFRACTION97
2.85-3.130.34631400.29491257X-RAY DIFFRACTION95
3.13-3.580.27231420.24871281X-RAY DIFFRACTION97
3.58-4.510.23831400.20511256X-RAY DIFFRACTION94
4.52-77.90.25561450.22221297X-RAY DIFFRACTION94
Refinement TLS params.Method: refined / Origin x: -7.8592 Å / Origin y: -8.7491 Å / Origin z: -19.7577 Å
111213212223313233
T0.4343 Å20.0011 Å2-0.1133 Å2-0.3826 Å2-0.017 Å2--0.4218 Å2
L1.4467 °20.1734 °2-1.9486 °2-0.7657 °2-0.067 °2--2.1568 °2
S-0.0826 Å °0.058 Å °0.0445 Å °-0.0299 Å °-0.0281 Å °-0.029 Å °0.0474 Å °-0.1082 Å °0.0872 Å °
Refinement TLS groupSelection details: all

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