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- PDB-7sw6: MicroED structure of proteinase K from a 260 nm thick lamella mea... -

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Basic information

Entry
Database: PDB / ID: 7sw6
TitleMicroED structure of proteinase K from a 260 nm thick lamella measured at 200 kV
ComponentsProteinase K
KeywordsHYDROLASE / Serine protease
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 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 ...Proteinase K-like catalytic 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
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 1.95 Å
AuthorsMartynowycz, M.W. / Clabbers, M.T.B. / Unge, J. / Hattne, J. / 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: Proc Natl Acad Sci U S A / Year: 2021
Title: Benchmarking the ideal sample thickness in cryo-EM.
Authors: Michael W Martynowycz / Max T B Clabbers / Johan Unge / Johan Hattne / Tamir Gonen /
Abstract: The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K ...The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.
History
DepositionNov 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proteinase K


Theoretical massNumber of molelcules
Total (without water)28,9311
Polymers28,9311
Non-polymers00
Water2,288127
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)67.910, 67.910, 102.520
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
IDModelComponents
11A-475-

HOH

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Components

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


Mass: 28930.783 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 127 / 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: 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 / Details: Microcrystals
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 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: 1 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 120 / Num. of grids imaged: 1 / Num. of real images: 1
Details: 0.5 degrees per second, 1 second readout, 30 to -30 degrees.
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1863 mm
EM diffraction shellResolution: 1.95→2.07 Å / Fourier space coverage: 92 % / Multiplicity: 5 / Num. of structure factors: 2561 / Phase residual: 29 °
EM diffraction statsFourier space coverage: 91 % / High resolution: 1.95 Å / Num. of intensities measured: 88107 / Num. of structure factors: 16540 / Phase error: 22 ° / Phase error rejection criteria: None / Rmerge: 28 / Rsym: 13
DetectorDate: Nov 14, 2020
ReflectionResolution: 1.95→40.91 Å / Num. obs: 88107 / % possible obs: 91.1 % / Redundancy: 5.3 % / Biso Wilson estimate: 22.29 Å2 / CC1/2: 0.976 / Rmerge(I) obs: 0.277 / Rpim(I) all: 0.133 / Rrim(I) all: 0.309 / Net I/σ(I): 4.25
Reflection shellResolution: 1.95→2 Å / Num. unique obs: 6241 / CC1/2: 0.411 / Rrim(I) all: 1.514 / Net I/σ(I) obs: 0.9 / % possible all: 92.7

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Processing

Software
NameVersionClassificationNB
PHENIX1.18.2_3874refinement
XDSdata reduction
XSCALEdata scaling
PHASER2.8.3phasing
EM software
IDNameCategory
6Cootmodel fitting
11AIMLESScrystallography merging
12PHENIX3D reconstruction
13PHENIXmodel refinement
Image processingDetails: Binned by 2.
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.91 Å / B: 67.91 Å / C: 102.52 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 20 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum likelihood
Atomic model buildingPDB-ID: 6CL7

6cl7


Pdb chain-ID: A / Accession code: 6CL7 / Pdb chain residue range: 106-384 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6CL7

6cl7


Resolution: 1.95→30.53 Å / SU ML: 0.2566 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 22.3984
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2385 798 4.82 %
Rwork0.1886 15742 -
obs0.191 16540 91.31 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 20.03 Å2
Refinement stepCycle: LAST / Resolution: 1.95→30.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2029 0 0 127 2156
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00952068
ELECTRON CRYSTALLOGRAPHYf_angle_d1.98142810
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0605312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0091370
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d10.6577307
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.95-2.070.34291360.26532561ELECTRON CRYSTALLOGRAPHY91.86
2.07-2.230.28591240.2422599ELECTRON CRYSTALLOGRAPHY91.84
2.23-2.460.29421440.22432588ELECTRON CRYSTALLOGRAPHY91.92
2.46-2.810.28341500.21382598ELECTRON CRYSTALLOGRAPHY91.66
2.81-3.540.22211320.17262646ELECTRON CRYSTALLOGRAPHY91.29
3.54-30.530.15591120.14282750ELECTRON CRYSTALLOGRAPHY89.41

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