STRUCTURE OF ACETYLCHOLINE RECEPTOR PORE FROM ELECTRON IMAGES
ELECTRON MICROSCOPY
Sample
POSTSYNAPTIC MEMBRANE
Specimen Preparation
Sample Aggregation State
FILAMENT
Vitrification Instrument
HOMEMADE PLUNGER
Cryogen Name
ETHANE
Sample Vitrification Details
LIQUID ETHANE
3D Reconstruction
Reconstruction Method
HELICAL
Number of Particles
Reported Resolution (Å)
4
Resolution Method
FSC 0.5 CUT-OFF
Other Details
NO REFINEMENT WAS CARRIED OUT ON THE MODEL. THE COORDINATES ARE PRELIMINARY. INCLUDED ARE ALL RESIDUES IN THE TRANSMEMBRANE SEGMENTS M1-M3 AND M4, AND ...
NO REFINEMENT WAS CARRIED OUT ON THE MODEL. THE COORDINATES ARE PRELIMINARY. INCLUDED ARE ALL RESIDUES IN THE TRANSMEMBRANE SEGMENTS M1-M3 AND M4, AND THE CONNECTING LINKS M1-M2 AND M2-M3. THE EXTENDED LOOP BETWEEN M3 AND M4, FORMING THE INTRACELLULAR DOMAIN OF THE RECEPTOR, AND SEVERAL OF THE C -TERMINAL RESIDUES ON M4 (CHAINS B, C AND E) ARE OMITTED. THE LINK BETWEEN M1 AND M2 WAS POORLY RESOLVED AND THE TRACE HERE IS ALMOST CERTAINLY WRONG IN DETAIL. IT IS HOPED THAT WITH IMPROVED DATA AND WITH REFINEMENT A MORE ACCURATE MODEL WILL BE OBTAINED. THESE COORDINATES, INCLUDING ESTIMATES OF SIDE CHAIN POSITIONS, ARE BEING MADE AVAILABLE IN THE HOPE THAT THEY WILL BE USEFUL. USERS SHOULD BEAR IN MIND THAT BECAUSE OF THE LIMITED RESOLUTION THE CONFORMATIONS OF THE SIDE CHAINS AND THEIR ATOMIC COORDINATES ARE NOT INDIVIDUALLY RELIABLE. ALSO THE ENDS OF THE HELICES ARE UNCERTAIN BY AT LEAST ONE RESIDUE. THE SURFACE LATTICE OF THE TUBULAR CRYSTALS HAS P2 SYMMETRY. THE FOUR HELICAL FAMILIES, DEFINED BY THE NUMBERS OF THE TWO PRINCIPAL HELICES REQUIRED TO FILL 360 DEGREES OF AZIMUTH, WERE: (-16,6),(-18,6),(-17,5),(-15,7). NEGATIVE INDICATES LEFT-HANDED. TO COMPARE AND COMBINE FOURIER TERMS FROM WITHIN A FAMILY IT WAS USUALLY NECESSARY TO REASSIGN LAYER-LINES TO MATCH THOSE CORRESPONDING TO A STANDARD HELICAL SELECTION RULE DEFINING A TYPICAL SET OF POSITIONS IN THE TRANSFORM. THE STANDARD SELECTION RULES WERE: L = 113N' + 314M (N = 2N') (-16,6) L = 25N' + 112M (N = 6N') (-18,6) L = 247N + 605M (-17,5) L = -91N + 662M (-15,7) WHERE L IS THE LAYER-LINE NUMBER, M IS AN INTEGER, N IS THE START NUMBER (I.E. NUMBER AROUND THE CIRCUMFERENCE) OF THE CONTRIBUTING HELIX, AND N=2N' INDICATES THAT THE START NUMBERS OCCUR IN MULTIPLES OF 2.
Refinement Type
Symmetry Type
HELICAL
Data Acquisition
Detector Type
KODAK SO-163 FILM
Electron Dose (electrons/Å**2)
20
Imaging Experiment
1
Date of Experiment
2002-10-01
Temperature (Kelvin)
4.2
Microscope Model
JEOL 3000SFF
Minimum Defocus (nm)
800
Maximum Defocus (nm)
1800
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS
1.3
Imaging Mode
BRIGHT FIELD
Specimen Holder Model
Nominal Magnification
40000
Calibrated Magnification
36800
Source
FIELD EMISSION GUN
Acceleration Voltage (kV)
300
Imaging Details
THE SPECIMENS WERE TUBULAR CRYSTALS GROWN IN LOW SALT BUFFER FROM ISOLATED TORPEDO POSTSYNAPTIC MEMBRANES. THEY WERE APPLIED TO THE THE MICROSCOPE GRIDS AND FROZEN IN THE SAME SOLUTION. THESE CRYSTALS WERE TOO SMALL AND DISTORTED TO YIELD MEANINGFUL ELECTRON DIFFRACTION PATTERNS, SO BOTH THE AMPLITUDE AND THE PHASE TERMS HAD TO BE MEASURED FROM FOURIER TRANSFORMS OF THE IMAGES. THE IMAGES WERE RECORDED USING A DOSE OF 2000 ELECTRONS/NM2 DURING THE PERIOD: JAN-1996 TO OCT-2002. THE DATASETS INVOLVED 4 HELICAL FAMILIES OF TUBES, WHICH WERE ANALYSED (INCLUDING CORRECTIONS FOR DISTORTIONS) AS DESCRIBED IN THE REFERENCES. STRUCTURES WERE SYNTHESISED FROM THE AMPLITUDE AND PHASE TERMS DERIVED FROM EACH HELICAL FAMILY. THE FINAL DATASET WAS OBTAINED BY AVERAGING THESE 4 STRUCTURES IN REAL SPACE.
