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- PDB-8gl3: De novo design of monomeric helical bundles for pH-controlled mem... -

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Basic information

Entry
Database: PDB / ID: 8gl3
TitleDe novo design of monomeric helical bundles for pH-controlled membrane lysis
ComponentspRLB-519
KeywordsDE NOVO PROTEIN / protein design / pH-responsive / membrane lysis
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsGoldbach, N. / Bera, A.K. / Baker, D. / Kang, A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States) United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Protein Sci / Year: 2023
Title: De novo design of monomeric helical bundles for pH-controlled membrane lysis.
Authors: Nicolas Goldbach / Issa Benna / Basile I M Wicky / Jacob T Croft / Lauren Carter / Asim K Bera / Hannah Nguyen / Alex Kang / Banumathi Sankaran / Erin C Yang / Kelly K Lee / David Baker /
Abstract: Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane ...Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here, we describe the design of small monomeric proteins with buried histidine containing pH-responsive hydrogen bond networks and membrane permeating amphipathic helices. Of the 30 designs that were experimentally tested, all expressed in Escherichia coli, 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH-dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH-responsiveness and control lysis of model membranes within a very narrow range of pH (6.0-5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms.
History
DepositionMar 20, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Refinement description / Category: pdbx_initial_refinement_model / Item: _pdbx_initial_refinement_model.type
Revision 1.2Nov 8, 2023Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: pRLB-519


Theoretical massNumber of molelcules
Total (without water)27,5991
Polymers27,5991
Non-polymers00
Water55831
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)32.120, 44.703, 182.579
Angle α, β, γ (deg.)90.000, 90.430, 90.000
Int Tables number5
Space group name H-MI121
Space group name HallC2y(x,y,-x+z)
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z+1/2
#4: -x+1/2,y+1/2,-z+1/2

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Components

#1: Protein pRLB-519


Mass: 27599.225 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 31 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.4 Å3/Da / Density % sol: 48.2 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8 / Details: 0.2 M Sodium fluoride and 20% (w/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.99998 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 4, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.99998 Å / Relative weight: 1
ReflectionResolution: 2.3→45.64 Å / Num. obs: 11502 / % possible obs: 98.36 % / Redundancy: 3.3 % / Biso Wilson estimate: 23.78 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.082 / Rpim(I) all: 0.052 / Net I/σ(I): 10.5
Reflection shellResolution: 2.3→2.53 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.144 / Mean I/σ(I) obs: 7.39 / Num. unique obs: 2812 / CC1/2: 0.976 / Rpim(I) all: 0.091 / % possible all: 98.13

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
DIALSdata reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.3→45.64 Å / SU ML: 0.2172 / Cross valid method: FREE R-VALUE / σ(F): 1.42 / Phase error: 29.0929
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2836 622 5.41 %
Rwork0.238 10880 -
obs0.2404 11502 98.37 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 46.04 Å2
Refinement stepCycle: LAST / Resolution: 2.3→45.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1600 0 0 31 1631
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00161599
X-RAY DIFFRACTIONf_angle_d0.33682121
X-RAY DIFFRACTIONf_chiral_restr0.0264238
X-RAY DIFFRACTIONf_plane_restr0.0031285
X-RAY DIFFRACTIONf_dihedral_angle_d14.1537684
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.530.30891550.23782658X-RAY DIFFRACTION98.22
2.53-2.90.26351670.24322676X-RAY DIFFRACTION97.2
2.9-3.650.28581420.22572768X-RAY DIFFRACTION99.76
3.65-45.640.28341580.24532778X-RAY DIFFRACTION98.29
Refinement TLS params.Method: refined / Origin x: 8.879 Å / Origin y: -7.312 Å / Origin z: 36.152 Å
111213212223313233
T0.130626554671 Å2-0.00505251078471 Å20.0213812019374 Å2-0.175752578267 Å2-0.0606928837649 Å2--0.183556758209 Å2
L2.45387639114 °2-0.491229070996 °2-0.831135296045 °2-2.200495235 °2-1.34783690293 °2--2.72451328743 °2
S0.0750647158982 Å °0.457226911539 Å °-0.0930397423271 Å °-0.176446076106 Å °0.0472748410748 Å °-0.0303733315338 Å °0.0857918009659 Å °-0.593079074475 Å °-0.0401827860087 Å °
Refinement TLS groupSelection details: ( CHAIN A AND RESID 6:221 )

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