BIOCH 590: Biomacromolecules Spring 2010 !"#$%&'()*+,-(."-/$,00#1",23-( Copyright: Jianhan Chen 456(7&,'3,'(.3%'( 8( 9&/5#:%"-(#;(9<=(9#>?0%(@%0&A( C:%"(DEF(#;(G'#H'(!"#$%&'(I$">5$>"%/(;"#J()*",-( First X-ray protein structures Hemoglobin and myoglobin, by Max Perutz and Sir John Kendrew, Discovery of DNA Structure and respectively, in 1958 Watson, J. D., & Crick, F. H. C. A structure for Function: Watson and Crick, L. A. Source: PDB deoxyribose nucleic acid. Nature 171, 737–738 (1953) Pray, Nature Education 1 (2008). 456(7&,'3,'(.3%'( B( 456(7&,'3,'(.3%'( K( !"#$%&'(I$">5$>"%(9%$%"J&',L#'(?-()*",-( @&13*$3"#>132>$(2"#$%&'(5"-/$,00#1",23-N( ."-/$,00#1",23-( !"#$%&%'()*+)',-+./$$*0/1"&) • !"#$%&'(2>"&M5,L#'N(M"/$(/$%2(#;(,00(/$">5$>",0(O%$%"J&',L#'(%P#"$/( • !"#$%&'(."-/$,00&Q,L#'N($3%(J#/$(53,00%'1&'1(2,"$R( – !"#O>5%(H%00*#"O%"%O(2"#$%&'(J#'#*5"-/$,0/(H&$3#>$(,'-(&'50>/&#'(,'O(0,"1%( %'#>13($#(O&P",5$()*+,-(?%,J(( – S#"%(,"$($3,'(/5&%'5%N(0,"1%0-(&J2#//&?0%($#(2"%O&5$(5"-/$,00&Q,L#'(5#'O&L#'/( • ."-/$,0(J#>'L'1N($",'/;%"(#;(5"-/$,0/(;"#J(/#0>L#'($#(O%$%5$#"( – !"#2%"(,0&1'J%'$(5,'(&J2"#:%(5#J20%$%'%//( • 9&P",5L#'(O,$,(5#00%5L#'N(5%'$",0(?>$(/#J%H3,$(/$,'O,"O( – +%5#"O(J#'#53"#J,L5("%T%5L#'/(H3&0%("#$,L'1($3%(5"-/$,0( – S>0L*H,:%0%'1$3(O,$,(5#00%5L#'(#'(/-'53"#$"#'/( • 9,$,(2"#5%//&'1N(&'O%A&'1U(&'$%1",L#'(,'O(/5,0&'1( • I$">5$>"%(5#'/$">5L#'N(23,/%(2"#?0%J( – S#0%5>0,"("%20,5%J%'$V(@%,:-(,$#J(J%$3#O/(( • I$">5$>"%("%M'%J%'$(,'O(,',0-/&/N( – +%/#0>L#'U(+*;,5$#"U(H3,$(,?#>$(?&#0#1-RW( http://www.bfsc.leidenuniv.nl/teaching http://proteincrystallography.org/ & Wikipedia456(7&,'3,'(.3%'( E( ."-/$,00&Q,L#'(/2,5%(,'O(/5"%%'&'1N( Roderick MacKinnon Nobel Prize in Chemistry 2003 Z"#H&'1(."-/$,0(+%J,&'/([,"1%0-(,'(="$(( @#H(J>53(&/($##(J>53W( Y3%(/>55%//(",$%(#;(5"-/$,00&Q,L#'( ,22%,"/(?&J#O,0(R( Try over 300 conditions is probably enough to tell whether it will even crystallize? MacKinnon, Nature (2002). http://www.ruppweb.org/Xray/tutorial/High_Throughput_EMBL_full.htm KcsA (PDB: 1r3j) 456(7&,'3,'(.3%'( X( 456(7&,'3,'(.3%'( D( ."-/$,0N(=/-JJ%$"&5(\'&$(,'O(\'&$(.%00(( I-JJ%$"-( • ."-/$,0N(/#0&O/(H&$3(4$",'/0,L#',06("%2%,$/(#;(,(/-JJ%$"&5(J#L;(4>'&$(5%006( X(5"-/$,0(I-/$%J/( • ='(,/-JJ%$"&5(>'&$(&/($3%(/J,00%/$(>'&$(#;(:#0>J%($3,$(5#'$,&'/(,00(#;($3%( B8(2#&'$(1"#>2/(4/-JJ%$"-6( /$">5$>",0(&';#"J,L#'(,'O($3,$(?-(,220&5,L#'(#;($3%(/-JJ%$"-(#2%",L#'/( _K(0,`5%($-2%/(4/3,2%6( 5,'("%2"#O>5%($3%(>'&$(5%00]( ab(8Bc(/2,5%(1"#>2/( International symbol (Herman-Mauguin symbol): shortest symbols to general all symmetry elements e.g., P3121 P: lattice centering type (primitive) 31: symmetry of primary axis (trigonal c-axis) 2: symmetry of secondary axis (2 fold) 1: symmetry of tertiary axis Restriction of chirality: 65 space groups for biomolecules! See wikipedia for nice overview of crystal symmetry!) 456(7&,'3,'(.3%'( ^( 456(7&,'3,'(.3%'( _c( )*",-(9&P",5L#'( I-/$%J,L5(+#$,L#'($#(!"#?%(i'L"%(+%5&2"#5,0([,`5%( Incident scattered • 9%$%"J&'%O(?-(%0%5$"#'(O%'/&$-(43-O"#1%'/( A single recording of diffraction, i.e., a 0,"1%0-(&':&/&?0%R6( 2 2 2 single reflection, at a fixed crystal 2sin" h k l orientation only sample a limited number of = 2 + 2 + 2 • 9&P",5L#'N(J,A&J>J(,J20&$>O%/(H3%'(,00( # a b c reciprocal lattice points! /5,d%"%O(H,:%/(,"%(&'(23,/%]( Systematically rotating (a precisely aligned) • e",11f/([,HN(4_^_E(<#?%0(!"&Q%V(g[(e",11(H,/(8E($3%']6( crystal allows all reciprocal lattice to be sampled (precession imaging). 2dsin" = n# $2sin" /# = n /d ! – ='10%(#;(O&P",5L#'(&/(h>,'LQ%O( Intensities of the reflections vary dramatically, which is related to the types – +%5&2"#5,0("%0,L#'/3&2N(0,"1%"(/2,5&'1(0%,O/($#( and positions of atoms within the unit cell. ! /J,00%"(O&P",5L#'(,'10%/( • S&00%"(&'O&5%/N(423)(3)$6( – O%M'%($3%(;,J&0-(#;(0,`5%(20,'%/( 2sin" n h 2 k 2 l2 = = n 2 + 2 + 2 # dhkl a b c – :#'([,>%(ih>,L#'( – I5,d%"&'1(:%5$#"N(42j!U((j"U($j#6( Ewald Sphere (radius = 1/!) ! (4"%5&2"#5,0(/2,5%R6( 456(7&,'3,'(.3%'( __( http://xray0.princeton.edu/~phil/Facility/Guides/XrayDataCollection.html456(7&,'3,'(.3%'( _8( +%0,L'1(9&P",5L#'(m'$%'/&L%/(,'O(=$#J&5(( 9&P",5L#'(!,d%"'(,'O(I2,5%(Z"#>2( .##"O&',$%/N(I$">5$>"%(k,5$#"/( • k>00-(,>$#J,L5W( • S>0L20%(,$#J/(H&$3&'($3%(>'&$(5%00(O&P",5$(,$($3%(/,J%(,'10%/(4e",11f/( • !%,l(2&5l&'1( [,H6U(?>$(H&$3(O&P%"%'$(,J20&$>O%/(,'O(23,/%/( • O&P",5L#'(2,d%"'( • I$">5$>",0(;,5$#"N(/>J(#;(/5,d%"(H,:%/(#;(,00(,$#J/(( N "%5#1'&L#'(4/-/$%J,L5( 2"i(hX j +kY j +lZ j ) fj: atomic scattering factor F(h,k,l) = f j e (X ,Y ,Z ): fractional cell coordinates ,?/%'5%/("%:%,0/(/2,5%( # j j j j =1 2 1"#>26( • I5,d%"%O(&'$%'/&L%/(,5$>,00-(#?/%":%ON( I(h,k,l) = F(h,k,l) • ."-/$,0(>'&$(5%00( – m';#"J,L#'(,?#>$(23,/%(&/(0#/$R( J#"23#0#1-( • I$">5$>",0(5,05>0,L#'(;"#J()*",-(O&P",5L#'N(,'(&':%"/%(2"#?0%J( 4O&J%'/&#'/6( ! – O%$%"J&',L#'( Y3%(;#"H,"O(2"#?0%J(#;(5,05>0,L'1($3%(O&P",5L#'(2,d%"'U(! 4523(3$63(;"#J(,$#J&5( 5##"O&',$%/(&/($"&:&,0( • .#J20%$%'%//( – Y3%(&':%"/%(2"#5%//(#;(5,05>0,L'1(,$#J&5(/$">5$>"%U(4783983:86U(&/(<CYU(,/($3%( 5"&L5,0(23,/%(&';#"J,L#'(&/(0#/$(4&]%]U($3%(;,J#>/(23,/%(2"#?0%J6](( Indexing 1! • k#>"&%"($",'/;#"J(?%$H%%'("%,0(,'O("%5&2"#5,0(/2,5%/( 1 Cell lengths (Angstrom): 6.4232 7.3423 15.7870! "(h,k,l) = F(h,k,l)e#2$i(hX +kY +lZ ) Cell angles (degree): 95.8000 96.5907 102.5697! V % ": “the electron density” …! h,k,l amplitude (known) phase (unknown) http://renzresearch.com/Precognition/html/chpt2_index.htm456(7&,'3,'(.3%'( _B( 456(7&,'3,'(.3%'( _K( ! .#J20%$%'%//U(=55>",5-(,'O(+%/#0>L#'(( • 9&P",5L#'/(,$(0,"1%"(,'10%/U(&]%]U(3&13%"(3U(lU(0U("%h>&"%O(;#"(3&13%"("%/#0>L#'( 4kY(>'5%"$,&'$-(2"&'5&20%6( • S&//&'1(O&P",5L#'/(,//>J%O($#(?%(Q%"#(?-(O%;,>0$(&'($3%(/>JJ,L#'N( "%O>5%O(,55>",5-(4,'O("%O>5%O("%/#0>L#'6( 1 "(h,k,l) = %F(h,k,l)e#2$i(hX +kY +lZ ) V h,k,l ! #$ ~ 1/T Structural factor data F(h,k): inner circle marks 2A resolution. Including additional data up to the outer circle increases resolution to 1A (Fig 12.16 of Tinoco) 456(7&,'3,'(.3%'( _E( 456(7&,'3,'(.3%'( _n( I#0:&'1($3%(!3,/%(!"#?0%J( !3,/%jI$">5$>"%(+%M'%J%'$N(+*k,5$#"( • =(;>'O,J%'$,0(2"#?0%J(?>$(H&$3(%/$,?0&/3%O(/#0>L#'/( • 9&"%5$(J%$3#ON(/J,00(J#0%5>0%/(4o_G(,$#J/6(H&$3(3&13*"%/#0>L#'(O,$,(4p_(=6( • S#0%5>0,"("%20,5%J%'$N(&;(&'&L,0(/$">5$>",0(J#O%0/(5,'(?%(%/LJ,$%O(4%]1]U( $3"#>13(3#J#0#1-(J#O%0&'16U(!,d%"/#'(J%$3#O/(5,'(?%(>/%O($#(O%$%"J&'%O( $3%(#"&%'$,L#'(,'O($3>/(&'&L,0(1>%//(#;(23,/%]( • m/#J#"23#>/("%20,5%J%'$N(&'$"#O>5&'1(#'%(#"(J#"%(3%,:-(,$#J/(4H&$3(0,"1%( %0%5$"#'(O%'/&L%/6](.3,'1%/(&'(O&P",5L#'(&'$%'/&L%/(H&$3(,'O(H&$3#>$(3%,:-( ,$#J/(5,'(?%(>/%O($#(#?$,&'(&'&L,0(23,/%(%/LJ,L#'(4>/&'1(%&$3%"(O&"%5$(#"( !,d%"/#'(J%$3#O/6](Cq%'(bB(O%"&:,L:%/('%5%//,"-]( • ='#J,0#>/(O&P",5L#'(4S=9(,'O(I=96(( http://www.sci.sdsu.edu/TFrey/Bio750/Bio750X-Ray.html • =q%"(&'&L,0(O%$%"J&',L#'(#;(23,/%U("%/>0L'1(/$">5$>",0(J#O%0(&/(>/%O($#( "%M'%($3%(23,/%/(,'O(5,05>0,$%(&J2"#:%O(J#O%0/(&'(,'(&$%",L:%(;,/3&#']( • r>,0&$-(#;(M`'1(J%,/>"%O(?-(+*;,5$#"N( – ;;"%%N(5,05>0,$%O(;"#J(p_cF(#;(O,$,('#$( ( ((((((,0"%,O-(&'50>O%O(&'("%M'%J%'$](( A 2.6 Å SIR electron-density Phase probability for one reflection in a MIR experiment. – (a) One derivative. (b) Three derivatives. map with the final C% trace of +;"%%(p("%/#0>L#'(j(_c(4&]%]U(p(c]8(;#"(8(=(/$">5$>"%6( the structure superimposed. http://journals.iucr.org/d/issues/2003/11/00/ba5050/index.html456(7&,'3,'(.3%'( _X( 456(7&,'3,'(.3%'( _D( ."-/$,0(e*k,5$#"/( e*k,5$#"(,'O(.#';#"J,L#',0(k0%A&?&0&$-( • =0/#(l'#H'(,/($%J2%",$>"%(;,5$#"(4&'50>O%O(&'(!9e(M0%/6( e*;,5$#"(a(DujB(+SIk8( • k&$($#($3%(O,$,(&'(#"O%"($#(,55#>'$(;#"($3%(s?0>""&'%//s(#;($3%(%0%5$"#'( O%'/&$-(O>%($#($3%"J,0(J#L#']( • e*;,5$#"/(#;(/&O%53,&'/(4E($#(nc(t86($#(?%(0,"1%"(,'O(J#"%(:,"&,?0%($3,'( ?,5l?#'%(,$#J/(4E($#(BE(t86](( • 4\'"%0&,?0%6(&'O&5,L#'(#;(2"#$%&'(O-',J&5/W( bacterial phosphotriesterase (PTE) Correlation between loop 7 B factor and kcat in PTE variants Jackson et al, PNAS (2009) 456(7&,'3,'(.3%'( _^( 456(7&,'3,'(.3%'( 8c( PDB Molecule of the Month: @#H(,?#>$(?&#0#1-W( Enhanceosome • ."-/$,0(,"L;,5$/N(5"-/$,00&Q,L#'(5#'O&L#'/(,'O(5"-/$,0(2,5l&'1( • g3,$(,?#>$(O-',J&5/W( • I$">5$>"%(&/(v>/$(,(?%1&''&'1(w( • <S+(,'O()*",-(5"-/$,00#1",23-(&/(5#J20%J%'$,"-R( NMR X-ray crystallography short time scale, protein folding long time scale, static structure solution, purity single crystal, purity < 20kD, domain any size, domain, complex functional active site active or inactive atomic nuclei, chemical bonds electron density resolution limit 2-3.5Å resolution limit 1.5-3.5Å PDB: 1t2k, 2pi0, 2o6g and 2o61 Dynamics Transient interactions Panne et al, Embo J.