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- PDB-5k7t: MicroED structure of thermolysin at 2.5 A resolution -

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Basic information

Entry
Database: PDB / ID: 5k7t
TitleMicroED structure of thermolysin at 2.5 A resolution
ComponentsThermolysin
KeywordsHYDROLASE
Function / homology
Function and homology information


thermolysin / metalloendopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
Elastase; domain 1 - #10 / Elastase; domain 1 / PepSY domain / Peptidase propeptide and YPEB domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain ...Elastase; domain 1 - #10 / Elastase; domain 1 / PepSY domain / Peptidase propeptide and YPEB domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif / Neutral Protease Domain 2 / Neutral Protease; domain 2 / Peptidase M4/M1, CTD superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Roll / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ISOPROPYL ALCOHOL / Thermolysin
Similarity search - Component
Biological speciesBacillus thermoproteolyticus (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.5 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 1.4Feb 28, 2024Group: 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

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Structure visualization

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Assembly

Deposited unit
A: Thermolysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,7248
Polymers34,3601
Non-polymers3647
Water37821
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area960 Å2
ΔGint-76 kcal/mol
Surface area12440 Å2
MethodPISA
Unit cell
Length a, b, c (Å)92.065, 92.065, 128.498
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Thermolysin / Thermostable neutral proteinase


Mass: 34360.336 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus thermoproteolyticus (bacteria) / References: UniProt: P00800, thermolysin

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Non-polymers , 5 types, 28 molecules

#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#5: Chemical ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL


Mass: 60.095 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / 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: Thermolysin / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.034634 MDa / Experimental value: NO
Source (natural)Organism: Bacillus thermoproteolyticus (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
145 %dimethyl sulfoxide1
250 mMTris1
32.5 Mcesium chlorideCsCl1
SpecimenEmbedding 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
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: 721 / Num. of grids imaged: 3 / Num. of real images: 721
Image scansSampling size: 0.0311999992 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1750 mm
EM diffraction shellResolution: 2.5→2.75 Å / Fourier space coverage: 96.8 % / Multiplicity: 12.2 / Num. of structure factors: 2741 / Phase residual: 47.2 °
EM diffraction statsFourier space coverage: 58.5 % / High resolution: 1.6 Å / Num. of intensities measured: 224846 / Num. of structure factors: 25029 / Phase error: 26.44 ° / Phase residual: 44.76 ° / Phase error rejection criteria: 0 / Rmerge: 0.634 / Rsym: 0.634
ReflectionResolution: 1.6→30.14 Å / Num. all: 224846 / Num. obs: 25029 / % possible obs: 58.5 % / Redundancy: 9 % / Rmerge(I) obs: 0.634 / Rpim(I) all: 0.219 / Net I/σ(I): 4
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allNum. unique obsRpim(I) allNet I/σ(I) obs% possible all
1.6-1.631.80.7943141730.61618
8.63-30.149.60.20631183260.0718.295.7

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Processing

SoftwareName: PHENIX / Version: (1.10_2155: ???) / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EM-Menu4.0.9.75image acquisition
5iMosflm/MOSFLM7.2.1diffraction indexing
6MOLREP11.4.05model fitting
8PHENIX1.10_2155model refinement
9MOLREP11.4.05molecular replacementStarting model PDB ID 2tli
11POINTLESS1.10.21symmetry determination
12AIMLESS0.5.25crystallography merging
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 120 ° / A: 90.75 Å / B: 90.75 Å / C: 126.13 Å / Space group name: P6122 / Space group num: 178
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 2TLI

2tli


Pdb chain-ID: A / Accession code: 2TLI / Pdb chain residue range: 1-316 / Source name: PDB / Type: experimental model
RefinementResolution: 2.5→30.135 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / σ(F): 1.41 / Phase error: 26.44 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3096 589 5.2 %
Rwork0.2899 --
obs0.291 11327 96.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0032497
ELECTRON CRYSTALLOGRAPHYf_angle_d0.5093399
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d11.821418
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.042361
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.003448
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5004-2.75190.36641680.34782573ELECTRON CRYSTALLOGRAPHY97
2.7519-3.14970.31321360.32692646ELECTRON CRYSTALLOGRAPHY97
3.1497-3.96690.32491370.26812680ELECTRON CRYSTALLOGRAPHY97
3.9669-30.13760.23361480.22722839ELECTRON CRYSTALLOGRAPHY97

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