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- PDB-1fbk: CRYSTAL STRUCTURE OF CYTOPLASMICALLY OPEN CONFORMATION OF BACTERI... -

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Basic information

Entry
Database: PDB / ID: 1fbk
TitleCRYSTAL STRUCTURE OF CYTOPLASMICALLY OPEN CONFORMATION OF BACTERIORHODOPSIN
ComponentsBACTERIORHODOPSIN
KeywordsPROTON TRANSPORT / PROTON PUMP / MEMBRANE PROTEIN / RETINAL PROTEIN / TWO-DIMENSIONAL CRYSTAL ELECTRON DIFFRACTION / SINGLE CRYSTAL
Function / homology
Function and homology information


photoreceptor activity / phototransduction / proton transmembrane transport / monoatomic ion channel activity / plasma membrane
Similarity search - Function
Bacterial rhodopsins retinal binding site. / Bacterial rhodopsins signature 1. / Rhodopsin, retinal binding site / Bacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
RETINAL / Bacteriorhodopsin
Similarity search - Component
Biological speciesHalobacterium salinarum (Halophile)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 3.2 Å
AuthorsSubramaniam, S. / Henderson, R.
CitationJournal: Nature / Year: 2000
Title: Molecular mechanism of vectorial proton translocation by bacteriorhodopsin.
Authors: S Subramaniam / R Henderson /
Abstract: Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium ...Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium salinarum. The chromophore retinal is covalently attached to the protein via a protonated Schiff base. Upon illumination, retinal is isomerized. The Schiff base then releases a proton to the extracellular medium, and is subsequently reprotonated from the cytoplasm. An atomic model for bacteriorhodopsin was first determined by Henderson et al, and has been confirmed and extended by work in a number of laboratories in the last few years. Here we present an atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin. A 'switch' mechanism ensures the vectorial nature of pumping. First, retinal unbends, triggered by loss of the Schiff base proton, and second, a protein conformational change occurs. This conformational change, which we have determined by electron crystallography at atomic (3.2 A in-plane and 3.6 A vertical) resolution, is largely localized to helices F and G, and provides an 'opening' of the protein to protons on the cytoplasmic side of the membrane.
History
DepositionJul 15, 2000Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2000Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Oct 4, 2017Group: Data collection / Refinement description / Category: em_image_scans / software
Revision 1.4Jan 31, 2018Group: Experimental preparation / Category: exptl_crystal_grow
Item: _exptl_crystal_grow.pdbx_details / _exptl_crystal_grow.temp
Revision 1.5Nov 3, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Assembly

Deposited unit
A: BACTERIORHODOPSIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,9632
Polymers26,6781
Non-polymers2841
Water0
1
A: BACTERIORHODOPSIN
hetero molecules

A: BACTERIORHODOPSIN
hetero molecules

A: BACTERIORHODOPSIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,8886
Polymers80,0353
Non-polymers8533
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area6470 Å2
ΔGint-51 kcal/mol
Surface area27970 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)62.45, 62.45, 100.9
Angle α, β, γ (deg.)90, 90, 120
Int Tables number143
Space group name H-MP3
DetailsTrimer is present in vivo. The crystals contain trimers related by crystal symmetry P3.

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Components

#1: Protein BACTERIORHODOPSIN /


Mass: 26678.330 Da / Num. of mol.: 1 / Mutation: D96G,F171C,F219L / Source method: isolated from a natural source / Source: (natural) Halobacterium salinarum (Halophile) / References: UniProt: P02945
#2: Chemical ChemComp-RET / RETINAL / Retinal


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 402
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography
Crystal symmetry∠γ: 120 ° / A: 62.45 Å / B: 62.45 Å / C: 100.9 Å / Space group name H-M: P3

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Sample preparation

ComponentName: CYTOPLASMICALLY OPEN CONFORMATION OF BACTERIORHODOPSIN
Source: RECOMBINANT
Molecular weightValue: .027 MDa / Experimental value: NO
Source (natural)Organism: Halobacterium salinarum (Halophile)
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingMaterial: glucose or trehalose
Crystal growTemperature: 310 K / Method: naturally occurring in vivo / pH: 7
Details: crystals are increased in size by fusion and annealing using detergents, pH 7, naturally occurring in vivo, temperature 37K
Crystal grow
*PLUS
Temperature: 4 ℃ / pH: 5.6 / Method: unknown
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
118-23 mg/mlprotein11
20.5 %(w/v)beta-octylglucopyranoside11
34 %(w/v)benzamidine11
41.75 Msodium phosphate11
51.8-2.3 Mammonium sulfate1reservoir

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Data collection

MicroscopyModel: FEI/PHILIPS CM12 / Details: imaging date 1998-12-01
Electron gunIllumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingFilm or detector model: GENERIC CCD / Num. of diffraction images: 1000
DiffractionMean temperature: 93 K
Diffraction sourceSource: ELECTRON MICROSCOPE / Type: OTHER / Wavelength: 0.033
DetectorType: OTHER / Detector: CCD / Date: Dec 1, 1998
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthWavelength: 0.033 Å / Relative weight: 1
ReflectionResolution: 3.2→200 Å / Num. all: 7298 / Num. obs: 5668 / % possible obs: 77.7 % / Observed criterion σ(I): 0 / Rmerge(I) obs: 0.173

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Processing

SoftwareName: CNS / Version: 0.9 / Classification: refinement
Crystal symmetry∠γ: 120 ° / A: 62.45 Å / B: 62.45 Å / C: 100.9 Å / Space group name H-M: P3
3D reconstructionResolution: 3.2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Details: 402 patterns merged (merging R 17.3%) to generate set of lattice lines covering ~87% of reciprocal space, with resolution of 3.2 A in-plane and 3.6 A vertically [primary citation]
RefinementResolution: 3.2→200 Å / Stereochemistry target values: Engh & Huber
Details: Each diffraction pattern was automatically indexed, and the spot intensities were integrated either using a raster (for patterns recorded at specimen tilts less than 30 degrees) or using ...Details: Each diffraction pattern was automatically indexed, and the spot intensities were integrated either using a raster (for patterns recorded at specimen tilts less than 30 degrees) or using profile fitting (for patterns recorded at specimen tilts at or greater than 30 degrees, which represented about 80% of the total data set). Each pattern was then compared to the curves recorded for wild-type bacteriorhodopsin in glucose at -100 degrees C [Ceska and Henderson J. Mol. Biol. 213: 539-560 (1990)], and the relative proportions of the four different twins determined. This exercise was carried out with all four theoretically possible orientations of the crystal axes relative to the previous reference curves to ensure that the data were merged correctly. From the initial set of 486 patterns chosen, 286 minimally twinned diffraction patterns were selected in which the major twin proportion was greater than 0.8. These 286 patterns were merged using the wild-type lattice lines as a reference and lattice lines were fitted to the data to obtain an initial approximately merged set of lattice lines (merge #1) describing the structure of the triple mutant. The original set of 486 patterns was then merged against the new lattice curves to redetermine the twin proportions more accurately. The merging parameters for each crystal were inspected carefully again, and 84 crystals for which the major twin proportion was less than 0.70 were excluded from the data set. The remaining 402 substantially untwinned diffraction patterns were used to generate a new set of curves and the procedure repeated to create a stable set of lattice lines. The merged data were further improved by using an estimate of sigma values for each reflection, and by the inclusion of an individual weighting factor for each diffraction pattern using procedures described by Grigorieff and Henderson [Ultramicroscopy 60: 295-309 (1995)]. Two cycles of this refinement were carried out to obtain a final set of merged curves. The curves were sampled at 1/100 Angstroms (approximately twice the thickness of the membrane) to obtain a set of intensities at H,K,L values so that the data could be further processed with standard X-ray crystallographic programs. For the tilt angles used, the maximal possible theoretical completeness of the data set is ~87%. The completeness of our data is close to this limit up to 3.5 Angstroms. The completeness drops to 77.7 % when all of the data to 3.2 Angstroms is included.
RfactorNum. reflection% reflectionSelection details
Rfree0.321 610 -RANDOM
Rwork0.272 ---
all-7298 --
obs-5668 77.7 %-
Refinement stepCycle: LAST / Resolution: 3.2→200 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1726 0 20 0 1746
Refine LS restraints
Refine-IDTypeDev ideal
ELECTRON CRYSTALLOGRAPHYc_bond_d0.009
ELECTRON CRYSTALLOGRAPHYc_angle_deg1.4

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