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- PDB-6kj2: 200kV MicroED structure of FUS (37-42) SYSGYS solved from single ... -

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Basic information

Entry
Database: PDB / ID: 6kj2
Title200kV MicroED structure of FUS (37-42) SYSGYS solved from single crystal at 0.67 A
ComponentsRNA-binding protein FUS
KeywordsRNA BINDING PROTEIN / FUS / MicroED / Ultrahigh resolution
Function / homology
Function and homology information


membraneless organelle assembly / mRNA stabilization / intracellular membraneless organelle / regulation of RNA splicing / Processing of Capped Intron-Containing Pre-mRNA / postsynaptic cytosol / positive regulation of double-strand break repair via homologous recombination / presynaptic cytosol / mRNA Splicing - Major Pathway / RNA splicing ...membraneless organelle assembly / mRNA stabilization / intracellular membraneless organelle / regulation of RNA splicing / Processing of Capped Intron-Containing Pre-mRNA / postsynaptic cytosol / positive regulation of double-strand break repair via homologous recombination / presynaptic cytosol / mRNA Splicing - Major Pathway / RNA splicing / mRNA 3'-UTR binding / transcription coregulator activity / molecular condensate scaffold activity / protein homooligomerization / GABA-ergic synapse / amyloid fibril formation / transcription coactivator activity / chromatin binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / glutamatergic synapse / DNA binding / RNA binding / zinc ion binding / nucleoplasm / identical protein binding / nucleus
Similarity search - Function
TAF15/EWS/TLS family / Zinc finger domain / Zn-finger in Ran binding protein and others / Zinc finger RanBP2 type profile. / Zinc finger, RanBP2-type superfamily / Zinc finger RanBP2-type signature. / Zinc finger, RanBP2-type / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. ...TAF15/EWS/TLS family / Zinc finger domain / Zn-finger in Ran binding protein and others / Zinc finger RanBP2 type profile. / Zinc finger, RanBP2-type superfamily / Zinc finger RanBP2-type signature. / Zinc finger, RanBP2-type / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
RNA-binding protein FUS
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 0.67 Å
AuthorsZhou, H. / Luo, F. / Luo, Z. / Li, D. / Liu, C. / Li, X.
Funding support China, 4items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2016YFA0501902 China
Ministry of Science and Technology (China)2016YFA0501102 China
National Natural Science Foundation of China31570730 China
National Natural Science Foundation of China91853113 China
CitationJournal: Anal Chem / Year: 2019
Title: Programming Conventional Electron Microscopes for Solving Ultrahigh-Resolution Structures of Small and Macro-Molecules.
Authors: Heng Zhou / Feng Luo / Zhipu Luo / Dan Li / Cong Liu / Xueming Li /
Abstract: Microcrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of ...Microcrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of this technique are still limited by the special requirement for radiation-tolerated movie-mode camera and the lack of automated data collection methods. Herein, we develop a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with a single-frame CCD camera, which ensures not only the acquisition of ultrahigh-resolution diffraction data but also low cost in practice. This method renders the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ∼0.60 Å with unambiguous assignment of nearly all hydrogen atoms. The present work provides a widely applicable solution for routine structure determination of MicroED and demonstrates the capability of the low-end 120 kV microscope with a CCD camera in solving ultrahigh resolution structures of both organic compounds and biological macromolecules.
History
DepositionJul 20, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Data processing / Category: em_3d_reconstruction / Item: _em_3d_reconstruction.resolution
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: RNA-binding protein FUS


Theoretical massNumber of molelcules
Total (without water)6631
Polymers6631
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area890 Å2
Unit cell
Length a, b, c (Å)18.110, 4.949, 18.665
Angle α, β, γ (deg.)90.000, 90.060, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein/peptide RNA-binding protein FUS / FUS LC RAC1


Mass: 662.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P35637
#2: Water ChemComp-HOH / 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: FUS LC RAC1 / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Mar 16, 2018
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: GATAN ULTRASCAN 4000 (4k x 4k)
EM diffractionCamera length: 520 mm
EM diffraction shellResolution: 0.67→0.69 Å / Fourier space coverage: 35.97 % / Multiplicity: 1.99 / Num. of structure factors: 227 / Phase residual: 1 °
EM diffraction statsFourier space coverage: 58.77 % / High resolution: 0.67 Å / Num. of intensities measured: 9323 / Num. of structure factors: 3780 / Phase error: 40.93 ° / Phase residual: 40.93 ° / Phase error rejection criteria: 1 / Rmerge: 0.123 / Rsym: 0.123

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0232refinement
PDB_EXTRACT3.25data extraction
EM 3D crystal entity∠α: 90 ° / ∠β: 90.06 ° / ∠γ: 90 ° / A: 18.11 Å / B: 4.95 Å / C: 18.66 Å / Space group name: P1211 / Space group num: 4
CTF correctionType: NONE
3D reconstructionResolution: 0.67 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 0.67→6.5 Å / Cor.coef. Fo:Fc: 0.904 / Cor.coef. Fo:Fc free: 0.957 / SU B: 0.523 / SU ML: 0.017 / Cross valid method: THROUGHOUT / σ(F): 1.4 / ESU R: 0.024 / ESU R Free: 0.023
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.244 191 5.1 %RANDOM
Rwork0.2353 ---
obs0.2357 3589 58.67 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 3.82 Å2 / Biso min: 1.51 Å2
Baniso -1Baniso -2Baniso -3
1--0.06 Å20 Å2-0.35 Å2
2--0.15 Å20 Å2
3----0.09 Å2
Refinement stepCycle: final / Resolution: 0.67→6.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms47 0 0 3 50
Biso mean---2.57 -
Num. residues----6
LS refinement shellResolution: 0.67→0.705 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.429 24 -
Rwork0.332 340 -
obs--39.35 %

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