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- PDB-6cl7: 1.71 A MicroED structure of proteinase K at 0.86 e- / A^2 -

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Basic information

Entry
Database: PDB / ID: 6cl7
Title1.71 A MicroED structure of proteinase K at 0.86 e- / A^2
ComponentsProteinase K
KeywordsHYDROLASE
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/S53 domain / 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 ...Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8/S53 domain / 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 / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 1.71 Å
AuthorsHattne, J. / Shi, D. / Glynn, C. / Zee, C.-T. / Gallagher-Jones, M. / Martynowycz, M.W. / Rodriguez, J.A. / Gonen, T.
CitationJournal: Structure / Year: 2018
Title: Analysis of Global and Site-Specific Radiation Damage in Cryo-EM.
Authors: Johan Hattne / Dan Shi / Calina Glynn / Chih-Te Zee / Marcus Gallagher-Jones / Michael W Martynowycz / Jose A Rodriguez / Tamir Gonen /
Abstract: Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron ...Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.
History
DepositionMar 2, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 16, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 4, 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.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Proteinase K


Theoretical massNumber of molelcules
Total (without water)28,9311
Polymers28,9311
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: PISA
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area9850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.010, 67.010, 100.722
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: 28930.783 Da / Num. of mol.: 1 / Fragment: UNP residues 106-384 / Source method: isolated from a natural source / Source: (natural) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
Has protein modificationY

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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: all / Source: NATURAL
Molecular weightValue: 0.028888994 MDa / Experimental value: NO
Source (natural)Organism: Engyodontium album (fungus)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
11.2 MAmmonium sulfateN2H8SO41
20.1 MTrisC4H11NO31
SpecimenConc.: 25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 30 %
CrystalPreparation: electron diffraction

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5.1 sec. / Electron dose: 0.0357 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 289 / Num. of grids imaged: 1 / Num. of real images: 289
Image scansSampling size: 31.2 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1200 mm
EM diffraction shellResolution: 1.71→1.75 Å / Fourier space coverage: 78.1 % / Multiplicity: 4.9 / Num. of structure factors: 1441 / Phase residual: 60.13 °
EM diffraction statsFourier space coverage: 93.4 % / High resolution: 1.71 Å / Num. of intensities measured: 144666 / Num. of structure factors: 23822 / Phase error: 38.96 ° / Phase residual: 38.96 ° / Phase error rejection criteria: 0 / Rmerge: 0.388 / Rsym: 0.388
DetectorDate: Mar 7, 2016

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Processing

Software
NameVersionClassification
REFMAC5.8.0194refinement
MOSFLM7.1.0data reduction
Aimless0.5.32data scaling
MOLREP11.4.05phasing
EM software
IDNameVersionCategory
1EM-Menu4.0.9.75image acquisition
6MOLREP11.4.05model fitting
8MOLREP11.4.05molecular replacement
10POINTLESS1.11.3symmetry determination
11AIMLESS0.5.32crystallography merging
13REFMAC5.8.0194model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.0104 Å / B: 67.0104 Å / C: 100.722 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.71 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 6.613 / Protocol: OTHER / Space: RECIPROCAL / Details: Electron scattering factors
Atomic model buildingPDB-ID: 5I9S

5i9s


Pdb chain-ID: A / Accession code: 5I9S / Pdb chain residue range: 1-279 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 5I9S

5i9s


Resolution: 1.71→50.01 Å / Cor.coef. Fo:Fc: 0.919 / Cor.coef. Fo:Fc free: 0.896 / SU B: 4.046 / SU ML: 0.122 / Cross valid method: THROUGHOUT / ESU R: 0.15 / ESU R Free: 0.137 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.25345 1547 6.5 %RANDOM
Rwork0.22126 ---
obs0.22336 22242 92.95 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 6.613 Å2
Baniso -1Baniso -2Baniso -3
1--0.07 Å20 Å20 Å2
2---0.07 Å20 Å2
3---0.14 Å2
Refinement stepCycle: 1 / Total: 2029
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0190.0192070
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0330.021806
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.7611.9362814
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.09234188
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg6.7015278
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg36.67423.61483
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg12.60115299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg13.771512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.110.2312
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0080.022405
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02435
ELECTRON CRYSTALLOGRAPHYr_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_nbd_other
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined
ELECTRON CRYSTALLOGRAPHYr_nbtor_other
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_other
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_mcbond_it0.4910.6151115
ELECTRON CRYSTALLOGRAPHYr_mcbond_other0.490.6131114
ELECTRON CRYSTALLOGRAPHYr_mcangle_it0.8380.921392
ELECTRON CRYSTALLOGRAPHYr_mcangle_other0.8380.9221393
ELECTRON CRYSTALLOGRAPHYr_scbond_it0.8310.74955
ELECTRON CRYSTALLOGRAPHYr_scbond_other0.8310.741956
ELECTRON CRYSTALLOGRAPHYr_scangle_it
ELECTRON CRYSTALLOGRAPHYr_scangle_other1.4241.0631423
ELECTRON CRYSTALLOGRAPHYr_long_range_B_refined1.9047.5392503
ELECTRON CRYSTALLOGRAPHYr_long_range_B_other1.9047.5462504
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr
ELECTRON CRYSTALLOGRAPHYr_sphericity_free
ELECTRON CRYSTALLOGRAPHYr_sphericity_bonded
LS refinement shellResolution: 1.708→1.752 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.336 90 -
Rwork0.346 1351 -
obs--78.1 %

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