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- PDB-8t1n: Micro-ED Structure of a Novel Domain of Unknown Function Solved w... -

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Basic information

Entry
Database: PDB / ID: 8t1n
TitleMicro-ED Structure of a Novel Domain of Unknown Function Solved with AlphaFold
ComponentsDUF1842 domain-containing protein
KeywordsUNKNOWN FUNCTION / AlphaFold / Bacterial Protein / Domain of Unknown Function / Novel Fold / Micro-ED
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)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 3 Å
AuthorsMiller, J.E. / Cascio, D. / Sawaya, M.R. / Cannon, K.A. / Rodriguez, J.A. / 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
DepositionJun 2, 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
Water00
1
A: DUF1842 domain-containing protein


Theoretical massNumber of molelcules
Total (without water)23,3141
Polymers23,3141
Non-polymers00
Water0
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
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)40.580, 94.990, 101.530
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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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

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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: DUF1842 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Burkholderia pseudomallei (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMBis-Tris pH 5.5C8H19NO51
2100 mMAmmonium AcetateNH4CH3COO1
317 %PEG 100001
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Data collection

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 10 sec. / Electron dose: 2 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 81
Image scansWidth: 2048 / Height: 2048
EM diffraction shellResolution: 3→47.25 Å / Fourier space coverage: 58.8 % / Multiplicity: 8.23 / Num. of structure factors: 4846 / Phase residual: 0.1 °
EM diffraction statsFourier space coverage: 58.8 % / High resolution: 3 Å / Num. of intensities measured: 39900 / Num. of structure factors: 4846 / Phase error rejection criteria: not applicable / Rmerge: 36.5
DetectorDate: Nov 1, 2019
ReflectionHighest resolution: 3.02 Å / Num. obs: 4846 / % possible obs: 58.8 % / CC1/2: 0.912 / Rmerge(I) obs: 0.365 / Rrim(I) all: 0.386 / Net I/σ(I): 5.58 / Num. measured all: 39900
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)% possible obs (%)Rmerge(I) obsNum. measured obsNum. unique obsCC1/2Rrim(I) allNet I/σ(I) obs
3.02-3.144.20.44411002680.140.5012.51
3.1-3.1857.40.48517753280.1970.5333.14
3.18-3.2759.40.43721863250.7250.4713.88
3.27-3.3760.90.43931253470.7960.4644.79
3.37-3.4860.90.42627253150.660.4514.75
3.48-3.6159.70.44928893100.750.4715.25
3.61-3.74610.36528073050.8190.3845.87
3.74-3.960.30.39426062860.8270.4136.04
3.9-4.07610.37227712940.9120.3916.34
4.07-4.2760.40.31222342550.9620.3286.82
4.27-4.561.90.28726082740.9740.3017.26
4.5-4.7759.70.24519962340.9890.2587.71
4.77-5.161.50.28622312380.9710.37.95
5.1-5.5158.60.32218402050.9570.346.5
5.51-6.0360.10.50417712020.9150.5275.4
6.03-6.7561.10.47517641870.9330.4975.59
6.75-7.7959.20.42714001610.9520.4496.05
7.79-9.5460.10.43610881430.730.4666.05
9.54-13.4958.50.3957371130.9690.426.84
13.4945.50.244247560.8080.2816.85

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Processing

EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 40.58 Å / B: 94.99 Å / C: 101.53 Å / Space group name: P212121 / Space group num: 19
CTF correctionType: NONE
3D reconstructionResolution: 3 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8T0B

8t0b


Resolution: 3→3 Å / SU ML: 0.47 / Cross valid method: THROUGHOUT / σ(F): 1.39 / Phase error: 24.2 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3066 242 5.02 %
Rwork0.2834 --
obs0.2845 4823 58.8 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0141783
ELECTRON CRYSTALLOGRAPHYf_angle_d1.5922441
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d10.446587
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.109282
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.01313
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.02-3.80.34491160.32312189ELECTRON CRYSTALLOGRAPHY57
3.8-35.030.27981260.25412392ELECTRON CRYSTALLOGRAPHY60
Refinement TLS params.Method: refined / Origin x: 4.4667 Å / Origin y: 28.0391 Å / Origin z: -19.6696 Å
111213212223313233
T0.0919 Å20.1115 Å2-0.0923 Å2--0.1632 Å20.0544 Å2--0.0885 Å2
L0.0212 °2-0.0268 °2-0.0007 °2-0.2365 °2-0.1303 °2--0.1985 °2
S0.0677 Å °-0.0871 Å °-0.0752 Å °0.055 Å °0.0477 Å °0.1175 Å °-0.037 Å °-0.1404 Å °0.0519 Å °
Refinement TLS groupSelection details: all

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