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- PDB-9cip: MicroED structure of the C11 cysteine protease clostripain -

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Basic information

Entry
Database: PDB / ID: 9cip
TitleMicroED structure of the C11 cysteine protease clostripain
ComponentsClostripain
KeywordsHYDROLASE / protease / thiol protease
Function / homologyclostripain / Peptidase C11, Clostripain Clostridium species / Peptidase C11, clostripain / Clostripain family / cysteine-type endopeptidase activity / proteolysis / Clostripain
Function and homology information
Biological speciesHathewaya histolytica (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.5 Å
AuthorsRuma, Y.N. / Bu, G. / Hattne, J. / Gonen, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Struct Biol X / Year: 2024
Title: MicroED structure of the C11 cysteine protease clostripain.
Authors: Yasmeen N Ruma / Guanhong Bu / Johan Hattne / Tamir Gonen /
Abstract: Clostripain secreted from is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine- ...Clostripain secreted from is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.
History
DepositionJul 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Clostripain
B: Clostripain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,7729
Polymers119,6112
Non-polymers1617
Water1086
1
A: Clostripain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,8744
Polymers59,8051
Non-polymers693
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Clostripain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,8975
Polymers59,8051
Non-polymers924
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)65.790, 106.070, 149.280
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121
Space group name HallP22ab(z,x,y)
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y+1/2,-z+1/2
#4: -x,-y+1/2,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 52 through 198 or resid 204 through 526))
d_2ens_1(chain "B" and (resid 52 through 452 or resid 460 through 526))

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11LYSLYSASNASNAA52 - 19852 - 198
d_12GLUGLUTRPTRPAA204 - 526204 - 526
d_21LYSLYSTYRTYRBB52 - 45252 - 452
d_22LYSLYSTRPTRPBB460 - 526460 - 526

NCS oper: (Code: givenMatrix: (-0.999998741723, 0.00113373839277, -0.00110959028168), (0.00113802435447, 0.999991865229, -0.00386967381433), (0.00110519405765, -0.00387093168597, -0.999991897184) ...NCS oper: (Code: given
Matrix: (-0.999998741723, 0.00113373839277, -0.00110959028168), (0.00113802435447, 0.999991865229, -0.00386967381433), (0.00110519405765, -0.00387093168597, -0.999991897184)
Vector: 32.9075540803, -0.0320863993283, 8.73909981471)

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Components

#1: Protein Clostripain


Mass: 59805.293 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Hathewaya histolytica (bacteria) / References: UniProt: P09870
#2: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Na
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: Clostripain / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.059 MDa / Experimental value: NO
Source (natural)Organism: Hathewaya histolytica (bacteria)
EM crystal formationTemperature: 293 K / Time: 1 DAY
Buffer solutionpH: 5.6
Buffer component
IDConc.NameFormulaBuffer-ID
10.2 MAmmonium acetateNH(4)CH(3)CO(2)1
20.1 MSodium-citrate tribasic dihydrate1
330 %PEG 40001
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 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 / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm
Specimen holderCryogen: NITROGEN / Temperature (max): 93 K / Temperature (min): 93 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 0.04 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 252 / Num. of grids imaged: 1 / Num. of real images: 252
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 20 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 2500 mm / Tilt angle list: -20,70
EM diffraction shellResolution: 2.5→2.56 Å / Fourier space coverage: 86.5 % / Multiplicity: 4.1 / Num. of structure factors: 2318 / Phase residual: 54.5 °
EM diffraction statsFourier space coverage: 86.8 % / High resolution: 2.5 Å / Num. of intensities measured: 131476 / Num. of structure factors: 32113 / Phase error: 34.49 ° / Phase error rejection criteria: 0 / Rmerge: 0.279
ReflectionBiso Wilson estimate: 29.39 Å2

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Processing

EM software
IDNameVersionCategory
1EPUimage acquisition
6PHENIX1.21.1_5286model fitting
8PHENIX1.21.1_5286model refinement
9PHENIX1.21.1_5287molecular replacement
12XSCALEJun 30, 2023crystallography merging
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 65.79 Å / B: 106.07 Å / C: 149.28 Å / Space group name: P22121 / Space group num: 18
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 24.7 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL
Atomic model buildingAccession code: A0A4U9RR22 / Source name: AlphaFold / Type: in silico model
RefinementResolution: 2.5→49.4 Å / SU ML: 0.3767 / Cross valid method: FREE R-VALUE / Phase error: 34.4948
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2726 1589 4.97 %
Rwork0.2379 30377 -
obs0.2396 31966 86.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 24.69 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00247481
ELECTRON CRYSTALLOGRAPHYf_angle_d0.546110102
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.04261038
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.00411332
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d13.48712754
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 0.381708976931 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.580.37571360.35082699ELECTRON CRYSTALLOGRAPHY85.86
2.58-2.670.34671430.33582742ELECTRON CRYSTALLOGRAPHY87.53
2.67-2.780.41261380.31762774ELECTRON CRYSTALLOGRAPHY87.34
2.78-2.910.3411360.30842752ELECTRON CRYSTALLOGRAPHY87.49
2.91-3.060.34181420.30722763ELECTRON CRYSTALLOGRAPHY87.71
3.06-3.250.3491440.27732768ELECTRON CRYSTALLOGRAPHY87.32
3.25-3.50.30171390.23872766ELECTRON CRYSTALLOGRAPHY87.34
3.5-3.850.21511770.19912738ELECTRON CRYSTALLOGRAPHY86.96
3.85-4.410.21721430.1742803ELECTRON CRYSTALLOGRAPHY86.65
4.41-5.560.17611430.16782802ELECTRON CRYSTALLOGRAPHY86.31
5.56-49.350.24291480.2122770ELECTRON CRYSTALLOGRAPHY81.85

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