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- PDB-6cla: 2.80 A MicroED structure of proteinase K at 6.0 e- / A^2 -

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Basic information

Entry
Database: PDB / ID: 6cla
Title2.80 A MicroED structure of proteinase K at 6.0 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/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 speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / FOURIER SYNTHESIS / cryo EM / Resolution: 2.8 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.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Proteinase K


Theoretical massNumber of molelcules
Total (without water)28,9311
Polymers28,9311
Non-polymers00
Water0
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 area9970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.450, 67.450, 100.818
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

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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: 240 / Num. of grids imaged: 1 / Num. of real images: 240
Image scansSampling size: 31.2 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1200 mm
EM diffraction shellResolution: 2.8→2.87 Å / Fourier space coverage: 88.21 % / Multiplicity: 5.8 / Num. of structure factors: 374 / Phase residual: 71.28 °
EM diffraction statsFourier space coverage: 88.4 % / High resolution: 2.8 Å / Num. of intensities measured: 30596 / Num. of structure factors: 5423 / Phase error: 62.59 ° / Phase residual: 62.59 ° / Phase error rejection criteria: 0 / Rmerge: 0.536 / Rsym: 0.536
DetectorDate: Mar 7, 2016

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Processing

Software
NameVersionClassification
REFMAC5.8.0194refinement
MOSFLM7.1.0data reduction
Aimless0.5.32data scaling
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: 67.4502 Å / B: 67.4502 Å / C: 100.818 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 2.8 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 29.658 / 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: FOURIER SYNTHESIS
Starting model: PDB entry 6CL7

6cl7


Resolution: 2.8→50.01 Å / Cor.coef. Fo:Fc: 0.875 / Cor.coef. Fo:Fc free: 0.773 / SU B: 38.53 / SU ML: 0.79 / Cross valid method: THROUGHOUT / ESU R Free: 0.618 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.32562 345 6.4 %RANDOM
Rwork0.26297 ---
obs0.26701 5039 87.69 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 29.658 Å2
Baniso -1Baniso -2Baniso -3
1-0.8 Å2-0 Å2-0 Å2
2--0.8 Å2-0 Å2
3----1.61 Å2
Refinement stepCycle: 1 / Total: 2029
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0120.0192070
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0740.021806
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.6781.9362814
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.21334188
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg8.35278
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg37.10423.61483
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg18.27815299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg16.0771512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0820.2312
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0060.022405
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0040.02435
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_it1.7363.021115
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.7373.0191114
ELECTRON CRYSTALLOGRAPHYr_mcangle_it2.9994.5311392
ELECTRON CRYSTALLOGRAPHYr_mcangle_other2.9984.5311393
ELECTRON CRYSTALLOGRAPHYr_scbond_it1.6113.138955
ELECTRON CRYSTALLOGRAPHYr_scbond_other1.6113.138956
ELECTRON CRYSTALLOGRAPHYr_scangle_it
ELECTRON CRYSTALLOGRAPHYr_scangle_other2.7094.6451423
ELECTRON CRYSTALLOGRAPHYr_long_range_B_refined6.06437.892637
ELECTRON CRYSTALLOGRAPHYr_long_range_B_other6.06337.8972638
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr
ELECTRON CRYSTALLOGRAPHYr_sphericity_free
ELECTRON CRYSTALLOGRAPHYr_sphericity_bonded
LS refinement shellResolution: 2.8→2.873 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.397 24 -
Rwork0.401 350 -
obs--88.21 %

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