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- PDB-6n4u: MicroED structure of Proteinase K at 2.75A resolution from a sing... -

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Basic information

Entry
Database: PDB / ID: 6n4u
TitleMicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.
ComponentsProteinase K
KeywordsHYDROLASE / broad-spectrum serum proteinase
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
Proteinase K-like catalytic domain / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. ...Proteinase K-like catalytic domain / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesEngyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.75 Å
AuthorsMartynowycz, M.W. / Zhao, W. / Hattne, J. / Jensen, G.J. / Gonen, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35 GM122588 United States
CitationJournal: Structure / Year: 2019
Title: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size.
Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
History
DepositionNov 20, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 6, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1354
Polymers28,9591
Non-polymers1763
Water41423
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area260 Å2
ΔGint-21 kcal/mol
Surface area10120 Å2
Unit cell
Length a, b, c (Å)67.460, 67.460, 106.670
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Proteinase K / / Endopeptidase K / Tritirachium alkaline proteinase


Mass: 28958.791 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Engyodontium album (fungus) / Gene: PROK / Production host: Engyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 23 / 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: Proteinase K / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.02893 MDa / Experimental value: NO
Source (natural)Organism: Engyodontium album (fungus)
Buffer solutionpH: 7.5
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Proteinase K purchased from Sigma.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 273 K

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 0.04 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 100 / Num. of grids imaged: 1
Details: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds.
Image scansSampling size: 28 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 1700 mm
EM diffraction shellResolution: 2.75→3.1483 Å / Fourier space coverage: 88.23 % / Multiplicity: 2.9 / Num. of structure factors: 1831 / Phase residual: 28.11 °
EM diffraction statsFourier space coverage: 88.4 % / High resolution: 2.75 Å / Num. of intensities measured: 31013 / Num. of structure factors: 10807 / Phase error: 22.41 ° / Phase residual: 22.41 ° / Phase error rejection criteria: 0 / Rmerge: 0.47 / Rsym: 0.39

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Processing

SoftwareName: PHENIX / Version: (1.14_3260: ???) / Classification: refinement
EM software
IDNameVersionCategory
6PHENIX1.14model fitting
8PHENIX2.8.2molecular replacement
13PHENIX1.14model refinement
Image processingDetails: This was the new CetaD.
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.46 Å / B: 67.46 Å / C: 106.67 Å / Space group name: P43212 / Space group num: 96
CTF correctionDetails: NONE / Type: NONE
3D reconstructionResolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 21.1 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: R
Atomic model buildingPDB-ID: 5I9S

5i9s


Accession code: 5I9S / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6CL7

6cl7


Resolution: 2.75→43.546 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.25
RfactorNum. reflection% reflectionSelection details
Rfree0.2626 383 6.37 %From 5I9S
Rwork0.2377 ---
obs0.2393 6008 87.76 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0052072
ELECTRON CRYSTALLOGRAPHYf_angle_d0.6972818
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d14.908720
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.044312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.004370
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7504-3.14830.33621240.30321831ELECTRON CRYSTALLOGRAPHY89
3.1483-3.96610.2561260.24081870ELECTRON CRYSTALLOGRAPHY88
3.9661-43.55110.23311330.20471924ELECTRON CRYSTALLOGRAPHY87

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