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- PDB-7n2l: MicroED structure of a mutant mammalian prion segment -

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Basic information

Entry
Database: PDB / ID: 7n2l
TitleMicroED structure of a mutant mammalian prion segment
Componentsprion protein
KeywordsPROTEIN FIBRIL / prion / MicroED / amyloid
Biological speciessynthetic construct (others)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 0.9 Å
AuthorsFlores, M.D. / Richards, L.S. / Zee, C.T. / Glynn, C. / Gallagher-Jones, M. / Sawaya, M.R.
Funding support United States, Brazil, Spain, 13items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1548924 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128867 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136614 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM007185 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)AI143368 United States
Sao Paulo Research Foundation (FAPESP)16/24191-8 United States
Sao Paulo Research Foundation (FAPESP)17/13485-3 Brazil
Spanish Ministry of Science, Innovation, and UniversitiesBES-2015-071397 United States
Spanish Ministry of Economy and CompetitivenessPGC2018-101370-B-100, Spain
Spanish Ministry of Economy and CompetitivenessMDM2014-0435-01 Spain
Other government2017SGR- 1192 Spain
CitationJournal: ACS Bio Med Chem Au / Year: 2023
Title: Fragment-Based Phasing of Peptidic Nanocrystals by MicroED.
Authors: Logan S Richards / Maria D Flores / Claudia Millán / Calina Glynn / Chih-Te Zee / Michael R Sawaya / Marcus Gallagher-Jones / Rafael J Borges / Isabel Usón / Jose A Rodriguez /
Abstract: Electron diffraction (MicroED/3DED) can render the three-dimensional atomic structures of molecules from previously unamenable samples. The approach has been particularly transformative for peptidic ...Electron diffraction (MicroED/3DED) can render the three-dimensional atomic structures of molecules from previously unamenable samples. The approach has been particularly transformative for peptidic structures, where MicroED has revealed novel structures of naturally occurring peptides, synthetic protein fragments, and peptide-based natural products. Despite its transformative potential, MicroED is beholden to the crystallographic phase problem, which challenges its determination of structures. ARCIMBOLDO, an automated, fragment-based approach to structure determination, eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptide structures including fragments of human amyloids, and yeast and mammalian prions. For electron diffraction, fragment-based phasing portends a more general phasing solution with limited model bias for a wider set of chemical structures.
History
DepositionMay 29, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 1, 2022Provider: repository / Type: Initial release
Revision 1.1May 10, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: prion protein


Theoretical massNumber of molelcules
Total (without water)1,1411
Polymers1,1411
Non-polymers00
Water362
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area1380 Å2
MethodPISA
Unit cell
Length a, b, c (Å)4.870, 10.060, 30.660
Angle α, β, γ (deg.)94.850, 90.260, 99.990
Int Tables number1
Space group name H-MP1

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Components

#1: Protein/peptide prion protein


Mass: 1141.147 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / 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: 0.9 A MicroED structure of a mutant mammalian prion segment
Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Buffer solutionpH: 4.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.05 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)
EM diffractionCamera length: 520 mm
EM diffraction shell
Resolution (Å)IDEM diffraction stats-IDFourier space coverage (%)MultiplicityNum. of structure factorsPhase residual (°)
7.9807-1.931211795.833500.01
1.932-1.53642180.45.83190.01
1.5364-1.34323180.75.863640.01
1.3432-1.22094180.55.423170.01
1.2209-1.13375181.75.53760.01
1.1337-1.0676183.35.323200.01
1.067-1.01377179.35.13600.01
1.0137-0.96968181.75.053030.01
0.9696-0.93239181.75.073980.01
0.9323-0.900210182.34.863300.01
EM diffraction statsFourier space coverage: 0.81 % / High resolution: 0.9 Å / Num. of intensities measured: 18502 / Num. of structure factors: 3437 / Phase error rejection criteria: 0.01 / Rmerge: 19.7
RadiationScattering type: electron
Radiation wavelengthRelative weight: 1
ReflectionResolution: 0.9→7.98 Å / Num. obs: 3437 / % possible obs: 81 % / Redundancy: 5.383 % / Biso Wilson estimate: 6.859 Å2 / CC1/2: 0.988 / Rmerge(I) obs: 0.197 / Rrim(I) all: 0.214 / Χ2: 0.824 / Net I/σ(I): 5.21 / Num. measured all: 18502 / Scaling rejects: 10
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
0.9-0.934.8580.4752.5616034013300.7830.52982.3
0.93-0.975.0680.3923.320174873980.8120.43581.7
0.97-1.015.0530.3893.2715313713030.8940.43281.7
1.01-1.075.1030.3094.1418374543600.9390.34279.3
1.07-1.135.3280.2824.9717053843200.9230.30983.3
1.13-1.225.50.2825.4920684603760.910.3181.7
1.22-1.345.4160.2725.6317173943170.9340.29980.5
1.34-1.545.8570.2466.6321324513640.9370.26780.7
1.54-1.935.7990.2037.5718503973190.9720.2280.4
1.93-7.985.8340.1518.6120424433500.9860.16379

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
XDSdata reduction
XSCALEdata scaling
PDB_EXTRACT3.27data extraction
EM 3D crystal entity∠α: 94.85 ° / ∠β: 90.264 ° / ∠γ: 99.988 ° / A: 4.87 Å / B: 10.06 Å / C: 30.66 Å / Space group name: P1 / Space group num: 1
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: AB INITIO MODEL
RefinementResolution: 0.9→7.98 Å / SU ML: 0.07 / Cross valid method: THROUGHOUT / σ(F): 2.13 / Phase error: 23.04 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.198 310 9.02 %RANDOM
Rwork0.1966 3125 --
obs0.1967 3435 81.09 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 28.82 Å2 / Biso mean: 5.2732 Å2 / Biso min: 0.5 Å2
Refinement stepCycle: final / Resolution: 0.9→7.98 Å
LigandSolventTotal
Num. atoms0 2 150
Biso mean-9.66 -
Num. residues--9
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Rfactor Rfree error: 0 / Total num. of bins used: 2

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
0.9-1.130.22151560.23121571172782
1.13-7.980.1881540.18181554170881

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