Journal: 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
Deposition
Jul 15, 2000
Deposition site: RCSB / Processing site: RCSB
Revision 1.0
Aug 9, 2000
Provider: repository / Type: Initial release
Revision 1.1
Apr 27, 2008
Group: Version format compliance
Revision 1.2
Jul 13, 2011
Group: Derived calculations / Version format compliance
Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O
-
Experimental details
-
Experiment
Experiment
Method: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 167
EM experiment
Aggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography
-
Sample preparation
Component
Name: Bacteriorhodopsin / Type: COMPLEX
Specimen
Embedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Crystal grow
Temperature: 310 K / Method: naturally occurring in vivo / pH: 7 Details: crystal size is increased 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
ID
Conc.
Common name
Crystal-ID
Sol-ID
1
18-23 mg/ml
protein
1
1
2
0.5 %(w/v)
beta-octylglucopyranoside
1
1
3
4 %(w/v)
benzamidine
1
1
4
1.75M
sodiumphosphate
1
1
5
1.8-2.3 M
ammoniumsulfate
1
reservoir
-
Data collection
EM imaging
Specimen-ID: 1
ID
Accelerating voltage (kV)
Details
Illumination mode
Model
Mode
Temperature (max) (K)
Cryogen
Nominal magnification (X)
Electron source
1
120
60degreetiltedspecimens
FLOODBEAM
FEI/PHILIPS EM420
DIFFRACTION
153
2
100
0, 20, 45degree + randomdegreetilts
FLOODBEAM
SIEMENS SULEIKA
BRIGHTFIELD
5
HELIUM
66000
3
100
, 20, 45degree + randomdegreetilts
SPOTSCAN
JEOL 100B
BRIGHTFIELD
158
NITROGEN
55000
FIELD EMISSION GUN
Image recording
ID
Imaging-ID
Average exposure time (sec.)
Electron dose (e/Å2)
Film or detector model
Num. of real images
Num. of diffraction images
2
2
12
20
GENERIC FILM
52
3
3
15
GENERIC FILM
20
1
1
GENERIC FILM
150
Diffraction
Mean temperature: 93 K
Diffraction source
Source: ELECTRON MICROSCOPE / Type: OTHER / Wavelength: 0.033
Detector
Type: OTHER / Detector: FILM / Date: Jan 1, 1986
Radiation
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Resolution: 3.2→200 Å / Stereochemistry target values: Engh & Huber Details: 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 ...Details: 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 65.1% when all of the data to 3.2 Angstroms is included.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.31
514
-
RANDOM
Rwork
0.239
-
-
-
all
-
7297
-
-
obs
-
4749
65.1 %
-
Refinement step
Cycle: LAST / Resolution: 3.2→200 Å
Protein
Nucleic acid
Ligand
Solvent
Total
Num. atoms
1733
0
20
0
1753
Refine LS restraints
Refine-ID
Type
Dev ideal
ELECTRONCRYSTALLOGRAPHY
c_bond_d
0.009
ELECTRONCRYSTALLOGRAPHY
c_angle_deg
1.4
+
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