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- PDB-7skx: Ab initio structure of proteinase K from electron-counted MicroED data -

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Entry
Database: PDB / ID: 7skx
TitleAb initio structure of proteinase K from electron-counted MicroED data
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
Chem-I3C / Proteinase K
Similarity search - Component
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.5 Å
AuthorsMartynowycz, M.W. / Clabbers, M.T.B. / 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: Nat Methods / Year: 2022
Title: Ab initio phasing macromolecular structures using electron-counted MicroED data.
Authors: Michael W Martynowycz / Max T B Clabbers / Johan Hattne / Tamir Gonen /
Abstract: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals ...Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.
History
DepositionOct 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 22, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,2498
Polymers28,9311
Non-polymers1,3187
Water4,216234
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)67.080, 67.080, 106.780
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11C-460-

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: 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.: 2 / Source method: obtained synthetically / Formula: C8H4I3NO4
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 234 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.028 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: Milled microcrystals
Specimen supportDetails: NEGATIVE / Grid 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: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Cs: 2.7 mm / C2 aperture diameter: 50 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: 0.5 sec. / Electron dose: 0.001 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 840 / Num. of grids imaged: 1 / Num. of real images: 1
Details: 0.15 degrees per second, 0.5 second readout, 30 to -30 degrees
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1738 mm / Tilt angle list: -30,30
EM diffraction shellResolution: 1.53→1.5 Å / Fourier space coverage: 89.3 % / Multiplicity: 8.9 / Num. of structure factors: 1758 / Phase residual: 30 °
EM diffraction statsDetails: Phases were determined by placing 4 ideal helix fragments. These were extended by chain tracing and density modifications.
Fourier space coverage: 98.87 % / High resolution: 1.5 Å / Num. of intensities measured: 416133 / Num. of structure factors: 39303 / Phase error: 20 ° / Phase error rejection criteria: None / Rmerge: 0.277 / Rsym: 0.087

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: 2020-24-08
Description: (un)restrained refinement or idealisation of macromolecular structures
EM software
IDNameCategory
8REFMACmodel refinement
12AIMLESScrystallography merging
13REFMAC3D reconstruction
Image processingDetails: Binned by 2.
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.08 Å / B: 67.08 Å / C: 106.78 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: 14.137 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood
RefinementResolution: 1.5→43.386 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.95 / SU B: 4.529 / SU ML: 0.067 / Cross valid method: FREE R-VALUE / ESU R: 0.085 / ESU R Free: 0.078
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflectionSelection details
Rfree0.2046 2001 5.091 %Random
Rwork0.1495 37302 --
all0.152 ---
obs-39303 98.848 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 14.137 Å2
Baniso -1Baniso -2Baniso -3
1--0.47 Å20 Å20 Å2
2---0.47 Å20 Å2
3---0.939 Å2
Refinement stepCycle: LAST / Resolution: 1.5→43.386 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2029 0 37 234 2300
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0160.0132102
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0010.0181858
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.6911.6372862
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.7211.5774266
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg6.7585278
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg34.94121.89595
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg12.28615299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg21.8371512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0830.2279
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0090.022497
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0010.02499
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2210.2554
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.2020.22047
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.1870.21155
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.090.21002
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1790.2218
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_other0.0980.21
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined0.0390.24
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.3330.220
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.2390.242
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.4050.217
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_other0.1270.22
ELECTRON CRYSTALLOGRAPHYr_mcbond_it1.8771.1631115
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.8581.161114
ELECTRON CRYSTALLOGRAPHYr_mcangle_it2.351.7531392
ELECTRON CRYSTALLOGRAPHYr_mcangle_other2.351.7571393
ELECTRON CRYSTALLOGRAPHYr_scbond_it3.6241.701987
ELECTRON CRYSTALLOGRAPHYr_scbond_other3.5891.683981
ELECTRON CRYSTALLOGRAPHYr_scangle_it4.4932.3981470
ELECTRON CRYSTALLOGRAPHYr_scangle_other4.4912.3991471
ELECTRON CRYSTALLOGRAPHYr_lrange_it5.13717.2642729
ELECTRON CRYSTALLOGRAPHYr_lrange_other4.91916.6342661
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr2.67933960
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.5390.3231560.3312501ELECTRON CRYSTALLOGRAPHY92.4817
1.539-1.5810.3291270.282673ELECTRON CRYSTALLOGRAPHY99.4318
1.581-1.6270.3031410.2382595ELECTRON CRYSTALLOGRAPHY99.7085
1.627-1.6770.2841200.2262538ELECTRON CRYSTALLOGRAPHY99.476
1.677-1.7320.2541410.1922413ELECTRON CRYSTALLOGRAPHY99.4161
1.732-1.7930.251240.1732360ELECTRON CRYSTALLOGRAPHY99.4794
1.793-1.8610.2371340.1542295ELECTRON CRYSTALLOGRAPHY99.4269
1.861-1.9370.2221220.1332187ELECTRON CRYSTALLOGRAPHY99.483
1.937-2.0230.2061140.1272120ELECTRON CRYSTALLOGRAPHY99.51
2.023-2.1210.1961100.1182034ELECTRON CRYSTALLOGRAPHY99.4896
2.121-2.2360.165900.1061950ELECTRON CRYSTALLOGRAPHY99.4637
2.236-2.3720.195890.1141847ELECTRON CRYSTALLOGRAPHY99.1295
2.372-2.5350.189910.1181720ELECTRON CRYSTALLOGRAPHY99.3963
2.535-2.7380.215840.1171627ELECTRON CRYSTALLOGRAPHY99.4189
2.738-2.9990.174890.1231492ELECTRON CRYSTALLOGRAPHY99.1844
2.999-3.3530.165750.1291370ELECTRON CRYSTALLOGRAPHY99.381
3.353-3.8710.15600.131208ELECTRON CRYSTALLOGRAPHY98.9852
3.871-4.740.136710.1291039ELECTRON CRYSTALLOGRAPHY98.9305
4.74-6.6960.159430.138834ELECTRON CRYSTALLOGRAPHY99.2081
6.696-43.380.374200.27499ELECTRON CRYSTALLOGRAPHY96.8284

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