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- PDB-5k7s: MicroED structure of proteinase K at 1.6 A resolution -

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Basic information

Entry
Database: PDB / ID: 5k7s
TitleMicroED structure of proteinase K at 1.6 A resolution
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 speciesEngyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.6 Å
Authorsde la Cruz, M.J. / Hattne, J. / Shi, D. / Seidler, P. / Rodriguez, J. / Reyes, F.E. / Sawaya, M.R. / Cascio, D. / Eisenberg, D. / Gonen, T.
CitationJournal: Nat Methods / Year: 2017
Title: Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.
Authors: M Jason de la Cruz / Johan Hattne / Dan Shi / Paul Seidler / Jose Rodriguez / Francis E Reyes / Michael R Sawaya / Duilio Cascio / Simon C Weiss / Sun Kyung Kim / Cynthia S Hinck / Andrew P ...Authors: M Jason de la Cruz / Johan Hattne / Dan Shi / Paul Seidler / Jose Rodriguez / Francis E Reyes / Michael R Sawaya / Duilio Cascio / Simon C Weiss / Sun Kyung Kim / Cynthia S Hinck / Andrew P Hinck / Guillermo Calero / David Eisenberg / Tamir Gonen /
Abstract: Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from ...Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.
History
DepositionMay 26, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2017Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2017Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.3Aug 22, 2018Group: Data collection / Database references / Category: pdbx_related_exp_data_set / Item: _pdbx_related_exp_data_set.data_reference
Revision 1.4Oct 23, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature / pdbx_struct_conn_angle / struct_conn
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 / _em_admin.last_update / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0113
Polymers28,9311
Non-polymers802
Water3,981221
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area90 Å2
ΔGint-12 kcal/mol
Surface area9990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.059, 67.059, 100.713
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-570-

HOH

21A-720-

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) 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 221 / Source method: isolated from a natural source / Formula: H2O
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: #1 / Source: NATURAL
Molecular weightValue: 0.028938 MDa / Experimental value: NO
Source (natural)Organism: Engyodontium album (fungus)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
11.2 Mammonium sulfateNH4SO41
20.1 MTris1
SpecimenConc.: 25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Mar 7, 2016
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.1 sec. / Electron dose: 0.004 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 295 / Num. of grids imaged: 1 / Num. of real images: 295
Image scansSampling size: 0.0311999992 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1200 mm
EM diffraction shellResolution: 1.6→1.64 Å / Fourier space coverage: 85.8 % / Multiplicity: 5.7 / Num. of structure factors: 1857 / Phase residual: 53.1 °
EM diffraction statsFourier space coverage: 81.1 % / High resolution: 1.3 Å / Num. of intensities measured: 302892 / Num. of structure factors: 46369 / Phase error: 24.33 ° / Phase residual: 32.2 ° / Phase error rejection criteria: 0 / Rmerge: 0.728 / Rsym: 0.728
ReflectionResolution: 1.3→20.75 Å / Num. all: 302892 / Num. obs: 46369 / % possible obs: 81.1 % / Redundancy: 6.5 % / Rmerge(I) obs: 0.728 / Rpim(I) all: 0.295 / Net I/σ(I): 2.4
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allNum. unique obsRpim(I) allNet I/σ(I) obs% possible all
1.3-1.3222.455204410051.9840.436.6
7.12-20.757.10.227393860.0738.193.1

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Processing

SoftwareName: PHENIX / Version: (1.10_2155: ???) / Classification: refinement
EM software
IDNameVersionCategory
1EM-Menu4.0.9.75image acquisition
5iMosflm/MOSFLM7.2.1diffraction indexing
6MOLREP11.4.05model fitting
8PHENIX1.10_2155model refinement
9MOLREP11.4.05molecular replacement
11POINTLESS1.10.21symmetry determination
12AIMLESS0.5.25crystallography merging
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.6 Å / B: 67.6 Å / C: 101.36 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.6 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
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
RefinementResolution: 1.6→20.751 Å / SU ML: 0.25 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 24.33
RfactorNum. reflection% reflection
Rfree0.2546 1936 6.5 %
Rwork0.2235 --
obs0.2255 29776 96.05 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0042070
ELECTRON CRYSTALLOGRAPHYf_angle_d0.6332814
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d9.171214
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.043312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.003370
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.640.42251240.36331733ELECTRON CRYSTALLOGRAPHY86
1.64-1.68440.38131280.34781818ELECTRON CRYSTALLOGRAPHY90
1.6844-1.73390.35091240.32991909ELECTRON CRYSTALLOGRAPHY94
1.7339-1.78980.32161320.29811998ELECTRON CRYSTALLOGRAPHY97
1.7898-1.85380.37081390.28071983ELECTRON CRYSTALLOGRAPHY98
1.8538-1.92790.30521420.25731997ELECTRON CRYSTALLOGRAPHY98
1.9279-2.01560.25931430.24432007ELECTRON CRYSTALLOGRAPHY98
2.0156-2.12180.26571430.22342010ELECTRON CRYSTALLOGRAPHY98
2.1218-2.25460.27241390.20962013ELECTRON CRYSTALLOGRAPHY98
2.2546-2.42840.25381460.21652018ELECTRON CRYSTALLOGRAPHY98
2.4284-2.67230.22431400.21312034ELECTRON CRYSTALLOGRAPHY98
2.6723-3.0580.24181420.19952066ELECTRON CRYSTALLOGRAPHY98
3.058-3.84870.16531420.15452081ELECTRON CRYSTALLOGRAPHY98
3.8487-20.75280.16331520.15092173ELECTRON CRYSTALLOGRAPHY97

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