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- PDB-7jsy: Proteinase K soaked with I3C determined by MicroED from a single ... -

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Basic information

Database: PDB / ID: 7jsy
TitleProteinase K soaked with I3C determined by MicroED from a single milled microcrystal
ComponentsProteinase K
KeywordsHYDROLASE / Proteinase K
Function / homology
Function and homology information

peptidase K / serine-type endopeptidase activity / metal ion binding
Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase S8, subtilisin-related / Peptidase S8, subtilisin, His-active site / Peptidase S8, subtilisin, Asp-active site / Peptidase S8, subtilisin, Ser-active site / Proteinase K-like catalytic domain / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily
Proteinase K
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.78 Å
AuthorsMartynowycz, M.W. / Gonen, T.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
CitationJournal: Structure / Year: 2020
Title: Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking.
Authors: Michael W Martynowycz / Tamir Gonen /
Abstract: A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy ...A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.
Validation Report
SummaryFull reportAbout validation report
DepositionAug 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release

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Deposited unit
A: Proteinase K
hetero molecules

Theoretical massNumber of molelcules
Total (without water)31,1665

TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area10650 Å2
Unit cell
Length a, b, c (Å)67.555, 67.555, 102.773
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2
Components on special symmetry positions



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

Mass: 28930.783 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical
ChemComp-I3C / 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid / 5-Amino-2,4,6-triiodoisophthalic acid

Mass: 558.835 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H4I3NO4
#3: Water ChemComp-HOH / water / Water

Mass: 18.015 Da / Num. of mol.: 246 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

Experimental details


EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

Sample preparation

ComponentName: Proteinase K / Type: COMPLEX / Details: Serine protease / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.0289 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

Data collection

MicroscopyModel: TFS TALOS
Details: Tilt series: -30 to +30, 0.25 deg/sec, 1 sec readout
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus min: 0 nm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 70 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 240 / Num. of grids imaged: 1 / Num. of real images: 240
Image scansSampling size: 28 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 1900 mm / Tilt angle list: -30,30
EM diffraction shellResolution: 43.32→1.78 Å / Fourier space coverage: 94.52 % / Multiplicity: 4.9 / Num. of structure factors: 10458 / Phase residual: 18 °
EM diffraction statsFourier space coverage: 94.52 % / High resolution: 1.78 Å / Num. of intensities measured: 109326 / Num. of structure factors: 10458 / Phase error: 18 ° / Phase residual: 18 ° / Phase error rejection criteria: 0 / Rmerge: 0.26 / Rsym: 0.13
ReflectionBiso Wilson estimate: 14.37 Å2


EM 3D crystal entityAngle alpha: 90 ° / Angle beta: 90 ° / Angle gamma: 90 ° / Length a: 67.55 Å / Length b: 67.55 Å / Length c: 102.77 Å / Space group name: 96 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.78 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 13.51 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: maximum liklihood
Atomic model buildingPDB-ID: 6CL7
Pdb chain-ID: A / Pdb chain residue range: 1-279
RefinementResolution: 1.78→43.32 Å / SU ML: 0.2175 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 19.6251
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2094 1449 6.53 %
Rwork0.1694 20756 -
Obs0.1721 22205 94.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 13.51 Å2
Refinement stepCycle: LAST / Resolution: 1.78→43.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2093 0 0 246 2339
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.045312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0038382
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d6.8398315
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefinement-ID% reflection obs (%)
1.78-1.840.35171400.28792033ELECTRON CRYSTALLOGRAPHY94.56
1.84-1.920.26051400.22852039ELECTRON CRYSTALLOGRAPHY95.15
1.92-20.25731540.19392037ELECTRON CRYSTALLOGRAPHY94.97
2-2.110.26311470.18312043ELECTRON CRYSTALLOGRAPHY94.93
2.11-2.240.22881360.17092059ELECTRON CRYSTALLOGRAPHY94.73
2.24-2.420.22541480.15512068ELECTRON CRYSTALLOGRAPHY94.62
2.42-2.660.22371460.16512049ELECTRON CRYSTALLOGRAPHY94.29
2.66-3.040.17771430.15642102ELECTRON CRYSTALLOGRAPHY94.61
3.04-3.830.16431410.14592103ELECTRON CRYSTALLOGRAPHY94.13

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