3J07

Model of a 24mer alphaB-crystallin multimer

SOLID-STATE NMR - SOLUTION SCATTERING - ELECTRON MICROSCOPY

Solution Scattering Data Acquistion	1
Scattering Type	x-ray
Radiation/Neutron Source	DESY HAMBURG X33 BEAMLINE
Synchrotron	Y
Beamline Type	X33
Detector Type	2D MAR345
Detector Manufacturer Details
Temperature (K)	293
pH	7.5
Numer of Time Frames Used	1
Protein Concentration Range (mg/mL)	0.1-12.6 mg/mL
Sample Buffer	phosphate, 100 mM NaCl
Data Reduction Software	CBF-PRIMUS
Guiner Mean Radius Of Gyration (nm)	5.6
Sigma Mean Radius Of Gyration	0.2
R(XS-1) Mean Cross Sectional Radii (nm)
R(XS-1) Sigma Mean Cross Sectional Radii
R(XS-2) Mean Cross Sectional Radii (nm)
R(XS-2) Sigma Mean Cross Sectional Radii
P(R) Protein Length (nm)	14.5

Solution Scattering Data Analysis and Model Fitting
Method	Software	Software Authors	Starting Model	Conformers, Number Calculated	Conformers, Number Submitted	Conformers, Selection Criteria	Best Representative Conformer	Other Details
FITTING OF MODELS WITH SAXS DATA;	CHIMERA, PRIMUS, GNOM, XPLOR-NIH, CRYSOL, DISCOVERY STUDIO 1.3;	PETOUKHOV, M.V.; SVERGUN, D.I.; SCHWIETERS, C.D. PETTERSON, E.F.; GODDARD, T.D.; ACCELRYS		10	1	BEST FIT WITH NMR, SAXS, AND EM DATA	1	TO DETERMINE HETEROGENEITY, A PYTHON SCRIPT WAS USED TO MODULATE AND FIT THE THEORETICAL SAXS CURVE OF THE MODEL WITH THE EXPERIMENTAL DATA. THE ALGOR ...	TO DETERMINE HETEROGENEITY, A PYTHON SCRIPT WAS USED TO MODULATE AND FIT THE THEORETICAL SAXS CURVE OF THE MODEL WITH THE EXPERIMENTAL DATA. THE ALGORITHM THAT WAS APPLIED HAS BEEN PUBLISHED BY MITTELBACH, R. AND GLATTER, O. (1998) IN J. APPL. CRYSTALLOGR. 31:600-608. FOR DETAILS SEE PRIMARY CITATION.

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 13C-13C PDSD	18 mg [1,3-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
2	2D 13C-13C PDSD	17 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
3	2D 13C-13C CHHC	4 mg [U-100% 13C; U-100% 15N] alphaB-crystallin, 16 mg alphaB-crystallin	100% H2O		7.6		270
4	2D 13C-13C PDSD	4 mg [U-100% 13C; U-100% 15N] alphaB-crystallin, 16 mg alphaB-crystallin	100% H2O		7.6		270
5	3D 15N-13C-13C NCACX	18 mg [1,3-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
6	3D 15N-13C-13C NCACX	17 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
7	3D 15N-13C-13C NCOCX	18 mg [1,3-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
8	3D 15N-13C-13C NCOCX	17 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
9	2D 15N-13C TEDOR	10 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
10	2D 15N-13C TEDOR	10 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [1,3-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
11	2D 15N-13C NHHC	10 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
12	2D 15N-13C NHHC	10 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [1,3-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
13	2D 15N-13C PAIN	10 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin	100% H2O		7.6		270
14	2D 1H-13C INEPT	4 mg [U-100% 13C; U-100% 15N] alphaB-crystallin, 16 mg alphaB-crystallin	100% H2O		7.6		270

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	Avance	400
2	Bruker	Avance	600
3	Bruker	Avance	700
4	Bruker	Avance	900

NMR Refinement
Method	Details	Software
simulated annealing, molecular dynamics, torsion angle dynamics	THREE DIMERS CONNECTED BY THEIR C-TERMINAL IXI MOTIF WERE FITTED BACK-TO-BACK IN THE EM DENSITY USING CHIMERA. A HEXAMER WAS BUILT FROM MODEL #7 OF PDB ENTRY 2KLR USING NONCRYSTALLOGRAPHIC SYMMETRY RESTRAINTS GIVEN IN THE PDB FILE. DIMERS WERE CONNECTED BY THEIR C-TERMINAL DOMAIN CONTAINING THE IXI-MOTIF, AND THE LINKER (UNP RESIDUES 151-155) WAS ENERGY MINIMIZED USING XPLOR-NIH. THE BETA1/BETA2(FREE) STRUCTURAL SEGMENT WAS PLACED INTO THE EM DENSITY TO FULFILL THE CROSSLINKS FOR A57, S59, AND T63, SIMILAR TO PDB ENTRY 1VLQ. ALPHA2 HELICES OF TWO MONOMERS WERE DOCKED, DEFINING RESIDUES L32, L33, L37, AND F38 AS INTERACTING RESIDUES USING HADDOCK. THE ALPHA2 HELICAL COMPLEX WAS PLACED IN WEAK EM DENSITY AT THE THREE-FOLD AXIS. THE FLEXIBLE C- AND N-TERMINAL RESIDUES, INCLUDING PARTS OF HELIX ALPHA1, WERE PLACED INSIDE THE 24MER TO FIT THE RADIUS OF GYRATION, MEASURED BY SMALL-ANGLE X-RAY SCATTERING IN AN ITERATIVE PROCESS. ALL FRAGMENTS WERE CONNECTED USING DISCOVERY STUDIO 1.6 (ACCELRYS) AND THE LINKERS WERE ENERGY MINIMIZED. BACK PROJECTIONS WERE CALCULATED FOR MODELS OF ALPHAB MULTIMERS CONTAINING 24, 26, AND 28 SUBUNITS USING THE PROGRAM SPIDER. THE CALCULATED PROJECTIONS ARE SIMILAR TO EACH OTHER, TO THE CLASS SUM IMAGES FROM COLLECTED EM DATA, AND TO THOSE CALCULATED FROM THE PUBLISHED EM DENSITY (EMD-1776). DETAILS ARE GIVEN IN THE PRIMARY CITATION.	CNS

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	200
Conformers Submitted Total Number	1
Representative Model	1 (lowest energy)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	collection	TopSpin		Bruker Biospin
2	processing	TopSpin		Bruker Biospin
3	chemical shift assignment	Sparky		Goddard
4	data analysis	Sparky		Goddard
5	peak picking	Sparky		Goddard
6	structure solution	X-PLOR NIH		Schwieters, Kuszewski, Tjandra and Clore
7	dihedral angle determination	TALOS		Cornilescu, Delaglio and Bax
8	structure solution	ARIA	2.2	Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M
9	refinement	ARIA	2.2	Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M
10	structure solution	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
11	refinement	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
12	structure solution	SOLARIA	1	(SOLARIA) Fossi M, Castellani F, Nilges M, Oschkinat H, van Rossum BJ

Sample
24mer alphaB-crystallin multimer

Specimen Preparation
Sample Aggregation State	PARTICLE
Staining Type	NEGATIVE
Staining Material	Uranyl Acetate
Staining Details

3D Reconstruction
Reconstruction Method	SINGLE PARTICLE
Number of Particles	2565
Reported Resolution (Å)	20
Resolution Method	FSC 0.5 CUT-OFF
Other Details
Refinement Type
Symmetry Type	POINT
Point Symmetry	T

Map-Model Fitting and Refinement
Id	1 (2KLR)
Refinement Space
Refinement Protocol
Refinement Target
Overall B Value
Fitting Procedure
Details

Data Acquisition
Detector Type	KODAK SO-163 FILM
Electron Dose (electrons/Å**2)

Imaging Experiment	1
Date of Experiment	2008-02-06
Temperature (Kelvin)
Microscope Model	JEOL 100CX
Minimum Defocus (nm)	900
Maximum Defocus (nm)	1200
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS
Imaging Mode	BRIGHT FIELD
Specimen Holder Model
Nominal Magnification	50000
Calibrated Magnification
Source	OTHER
Acceleration Voltage (kV)	100
Imaging Details

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.