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- PDB-9tbe: Cryo-EM structure of the light-driven sodium pump ErNaR in the mo... -

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Basic information

Entry
Database: PDB / ID: 9tbe
TitleCryo-EM structure of the light-driven sodium pump ErNaR in the monomeric form in the K2 state
ComponentsBacteriorhodopsin-like protein
KeywordsMEMBRANE PROTEIN / rhodopsin / ErNaR / sodium pump / phototransduction
Function / homologyEICOSANE / RETINAL
Function and homology information
Biological speciesErythrobacter (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHaubner, M. / Williams, H.M. / Hruby, J. / Straub, M.S. / Guskov, A. / Kovalev, K. / Drabbels, M. / Lorenz, U.J.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationTMCG-2+213773 Switzerland
CitationJournal: bioRxiv / Year: 2025
Title: Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification.
Authors: Michal Haubner / Harry M Williams / Jakub Hruby / Monique S Straub / Albert Guskov / Kirill Kovalev / Marcel Drabbels / Ulrich J Lorenz /
Abstract: Understanding and ultimately predicting the function of proteins is one of the frontiers in structural biology. This will only be possible if it becomes feasible to routinely observe proteins on the ...Understanding and ultimately predicting the function of proteins is one of the frontiers in structural biology. This will only be possible if it becomes feasible to routinely observe proteins on the fast timescales on which they perform their tasks. Recently, laser flash melting and revitrification experiments have improved the time resolution of cryo-electron microscopy (cryo-EM) to microseconds, rendering it fast enough to observe the domain motions of proteins that are frequently linked to function. However, observations have been limited to a time window of just a few hundred microseconds. Here, we introduce time-resolved cryo-EM experiments based on jet vitrification that combine microsecond resolution with an observation window of up to seconds. We use a short laser pulse to initiate protein dynamics, and as they unfold, vitrify the sample with a jet of a liquid cryogen to arrest the dynamics at that point in time. We demonstrate that our approach affords near-atomic spatial resolution and a time resolution of 21 μs. This allows us to observe the photoinduced dynamics of the light-driven sodium pump NaR on the microsecond to millisecond timescale. Our experiments significantly expand the ability of cryo-EM to observe protein dynamics across multiple timescales.
History
DepositionNov 19, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
D: Bacteriorhodopsin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,94615
Polymers30,5321
Non-polymers4,41414
Water36020
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Bacteriorhodopsin-like protein


Mass: 30532.338 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Erythrobacter (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical
ChemComp-LFA / EICOSANE / LIPID FRAGMENT


Mass: 282.547 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C20H42
#3: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#4: Chemical ChemComp-RET / RETINAL


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 20 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ErNaR light-driven sodium pump / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Erythrobacter (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 10 mM HEPES pH 7.5, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
210 mMTris-HClC4H11NO31
30.04 %n-dodecyl-beta-D-maltoside (DDM)C24H46O1
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportDetails: Grid was sputter coated with 40 nm thick layer of silver beforehand using a Quorum Q300T device.
Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: Vitrification carried out using a modified Vitrobot Mark IV, as outlined in the paper reporting this entry.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
4cryoSPARC4.6.0CTF correction
13cryoSPARC4.6.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44989
Details: Reconstruction conducted in C1 following symmetry expansion of particle stack in C5.
Symmetry type: POINT
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0042419
ELECTRON MICROSCOPYf_angle_d0.7983256
ELECTRON MICROSCOPYf_dihedral_angle_d12.011514
ELECTRON MICROSCOPYf_chiral_restr0.049357
ELECTRON MICROSCOPYf_plane_restr0.008384

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