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- PDB-5k7r: MicroED structure of trypsin at 1.7 A resolution -

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Basic information

Entry
Database: PDB / ID: 5k7r
TitleMicroED structure of trypsin at 1.7 A resolution
ComponentsCationic trypsin
KeywordsHYDROLASE
Function / homology
Function and homology information


trypsin / serpin family protein binding / serine protease inhibitor complex / digestion / endopeptidase activity / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding
Similarity search - Function
Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases ...Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / Thrombin, subunit H / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Biological speciesBos taurus (cattle)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.7 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

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Assembly

Deposited unit
A: Cationic trypsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,4043
Polymers23,3241
Non-polymers802
Water3,513195
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area170 Å2
ΔGint-21 kcal/mol
Surface area8620 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.184, 56.428, 64.672
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Cationic trypsin / Beta-trypsin


Mass: 23324.287 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P00760, trypsin
#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 / Water


Mass: 18.015 Da / Num. of mol.: 195 / 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: Trypsin / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.023354 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Buffer solutionpH: 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mg/mlbenzamidine1
23 mMcalcium chlorideCaCl1
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 / Date: Mar 11, 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: 1527 / Num. of grids imaged: 3 / Num. of real images: 1527
Image scansSampling size: 0.0311999992 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1500 mm
EM diffraction shellResolution: 1.7→1.79 Å / Fourier space coverage: 56.2 % / Multiplicity: 3.1 / Num. of structure factors: 1737 / Phase residual: 60.7 °
EM diffraction statsFourier space coverage: 73.8 % / High resolution: 1.5 Å / Num. of intensities measured: 145833 / Num. of structure factors: 23542 / Phase error: 28.86 ° / Phase residual: 40.4 ° / Phase error rejection criteria: 0 / Rmerge: 0.773 / Rsym: 0.773
ReflectionResolution: 1.5→27.63 Å / Num. all: 145833 / Num. obs: 23542 / % possible obs: 73.8 % / Redundancy: 6.2 % / Rmerge(I) obs: 0.773 / Rpim(I) all: 0.332 / Net I/σ(I): 2.3
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allNum. unique obsRpim(I) allNet I/σ(I) obs% possible all
1.5-1.531.43.6324022813.240.318.2
8.22-27.637.40.2116092170.0787.694.2

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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 2ptn
11POINTLESS1.10.21symmetry determination
12AIMLESS0.5.25crystallography merging
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 53.12 Å / B: 56.08 Å / C: 64.38 Å / Space group name: P212121 / Space group num: 19
CTF correctionType: NONE
3D reconstructionResolution: 1.7 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 2PTN

2ptn


Pdb chain-ID: A / Pdb chain residue range: 16-245
RefinementResolution: 1.7→25.86 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 28.86
RfactorNum. reflection% reflection
Rfree0.2811 919 4.77 %
Rwork0.2479 --
obs0.2495 19277 87.5 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0051666
ELECTRON CRYSTALLOGRAPHYf_angle_d0.7392264
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d12.1151000
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.049254
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.004289
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7001-1.78970.4237690.38721668ELECTRON CRYSTALLOGRAPHY56
1.7897-1.90180.43461110.36212261ELECTRON CRYSTALLOGRAPHY76
1.9018-2.04860.36061260.31652671ELECTRON CRYSTALLOGRAPHY90
2.0486-2.25460.33861410.26912884ELECTRON CRYSTALLOGRAPHY97
2.2546-2.58060.27151680.24292893ELECTRON CRYSTALLOGRAPHY97
2.5806-3.25030.23521730.21542916ELECTRON CRYSTALLOGRAPHY97
3.2503-25.86340.18041310.16643065ELECTRON CRYSTALLOGRAPHY97

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