Ford etal suppl subm#D63E50 · cedyn1/1-830 1 li n qd g lp vvg qs gkss le vg d lprg m pvi rv a fs l...

W W W. N A T U R E . C O M / N A T U R E | 1

SUPPLEMENTARY INFORMATIONdoi:10.1038/nature10441

NGTPase β1 α1

β2 β2A

αB β2B β3 α2

β4 α3 β5 α4 β6

P loopG1

Switch 1G2

Switch 2G3

G4

Transstabilizing

loopLL

Patch

ddDynA/1-853 1

rnDynI/1-864 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RL A FS I A LD QIA V N R F S I ..MGNRG D . A QN D G A F btDyn1/1-856 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RL A FS I A LD QIA V N R F S I ..MGNRG D . A QN D G A F hsDyn1/1-864 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RL A FS I A LD QIA V N R F S I ..MGNRG D . A QN D G A F mmDyn1/1-867 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RL A FS I A LD QIA V N R F S I ..MGNRG D . A QN D G A F ggDyn1/1-865 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RL A F I A LD QIA V N R F S I ..MGNRG D . AA QN N G A F drDyn1/1-843 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RM A FS I A LD QIA V N K F S I ..MGNRG D . A QN N G A F rnDyn2/1-870 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV KL A FS I LD QIA V N R F S I ..MGNRG E . S QSCH G A F rnDyn3/1-869 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV RL A FS L LE QIA V N R F S I ..MGNRE E . A QSCL G A F dmDyn/1-877 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR MD IV KL A FT L M LD QIA V N K F S I ....... S T . S VH Q G A F ceDyn1/1-830 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR M PVI RV A FS L V E QIA V N K F S I MSWQNQG QA . Q TS SF G A F

rnDrp1/1-755 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PVI KL V FN V I I QIV V S R T V ....... A . T AD . Q T S L L btDrp1/1-749 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PVI KL V FN V I I QIV V S R T I ....... A . T AD . Q T S L L hsDrp1/1-736 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PVI KL V FN V I I QIV V S R T I ....... A . T AD . Q T S L L mmDrp1/1-742 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PVI KL V FN V I I QIV V S R T V ....... A . T AD . Q T S L L drDrp1/1-691 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PVI KL V FN V I I QIA V S R T I ....... A . T AD . Q T S L L scDnm1/1-757 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR LE P V KL V Y LD LA I T R F T I ....MAS D T M DS. IDT. I S S L

spDnm1/1-781 1 LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR ME PLV L L YN I LD SIV V N K F T I ....... Q Q V T SDF. S C I LI N QD G LP VVG QS GKSS LE VG D LPRG G VTRR MD PVI KL V FN L LD QIV V N R F S I ....... Q . T SDP. S S I

rnDynI/1-864 68 PL LQL FLH V V EY E K K FTDFE N..........................................STT A C G K btDyn1/1-856 68 PL LQL FLH V V EY E K K FTDFE N..........................................ATT A C G K hsDyn1/1-864 68 PL LQL FLH V V EY E K K FTDFE N..........................................ATT A C G K mmDyn1/1-867 68 PL LQL FLH V V EY E K K FTDFE N..........................................STT A C G K ggDyn1/1-865 68 PL LQL FLH V V EY E K K FTDFE N..........................................AST G C G K drDyn1/1-843 68 PL LQL FLH V I EY E K K FTDFD N..........................................CPT A C G K rnDyn2/1-870 68 PL LQL FLH I I EY E K K FTDFD F..........................................SKT A C S K rnDyn3/1-869 68 PL LQL FLH V V EY E K K FTDFD T..........................................SKA A C G K dmDyn/1-877 63 PL LQL FLH I I EY E K K FS FD N..........................................GVT G I G K S ceDyn1/1-830 70 PL LQL FLH I I EY E K H F DFD Q..........................................DRN A K G R V

rnDrp1/1-755 62 PL LQL FLH I V EW K K YTDFD HVSPEDKRKTTGEENDPATWKNSRHLS.............KGVEAE GK T N L btDrp1/1-749 62 PL LQL FLH I V EW K K YTDFD HVAPEDKRKTTGEENDPATWKNSRHLS.............KGVEAE GK T N L hsDrp1/1-736 62 PL LQL FLH I V EW K K YTDFD HVSQEDKRKTTGEE..........................NGVEAE GK T N L mmDrp1/1-742 62 PL LQL FLH I V EW K K YTDFD HVSPEDKRKTTGEENGKFQS....................WRVEAE GK T N L drDrp1/1-691 62 PL LQL FLH I V EW K K YTDFD HVDPEDRRKTSEE...........................NGVDGE GK T N I scDnm1/1-757 65 PL LQL FLH V EW E K F DFD NNISPNSPLIEEDDNSVNPHDEVTKISGFEAGTKPLEYRGKERNHAD G IPG R Y

spDnm1/1-781 63 PL LQL FLH I I DY E FTD NLKRKTKNNHDEESTSDNNSEETSAAG.ETGS..LEGIEEDSDEIE A IPDTK MNddDynA/1-853 62 PL LQL FLH I EW E F DF THLPIAD..................................DGSQTQ G KPNDM Y S

rnDynI/1-864 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT TN IS N RVY P L V M VPV D IRDMLM FVT L A PV H P F Q btDyn1/1-856 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT TN IS N RVY P L V M VPV D IRDMLM FVT L A PV H P F Q hsDyn1/1-864 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT TN IS N RVY P L V M VPV D IRDMLM FVT L A PV H P F Q mmDyn1/1-867 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT TN IS N RVY P L V M VPV D IRDMLM FVT L A PV H P F Q ggDyn1/1-865 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E D VT SN IS N RVY P L V M VPV D IRDMLM FVT L A PV H P F Q drDyn1/1-843 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT QN IS N RVY P L V M VPV D IKDMLM FVT Q T PV N A H Q rnDyn2/1-870 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT TN IS N RVY P L I I VPV D IKDMIL FIS Q A PV H P Y Q rnDyn3/1-869 96 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EVR E E D VT N IS N RVY P L I I VPV D IRDMIM FIT H A M SV H P Y Q dmDyn/1-877 91 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E D VT SN IS N RVY P L I L V I D IK MI FI K D NI H A V Q Q FQ RceDyn1/1-830 98 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q VR E E D VT QN IS N RVF P L I L VPV D IRDMIL FINA K D PH N A Q T

rnDrp1/1-755 119 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E E IS NN VS KVF P V V M VPV D IRELIL FIS Q N PE H N K L R btDrp1/1-749 119 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E E IS NN VS KIF P V V M VPV D IRELIL FIS Q N PE H N K L R hsDrp1/1-736 106 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E E IS NN VS KIF P V V M VPV D IRELIL FIS Q N PE H N K L R mmDrp1/1-742 112 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E E IS NN VS KVF P V V M VPV D IRELIL FIS Q N PE H N K L R drDrp1/1-691 105 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR E E E VS NN IS KIF P V V I VPV D IRELIL YIS Q N DE H H K L K scDnm1/1-757 135 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q DIK E E I IS N KVF P L V I VPI E IK LIL YI R N A A KD KI H P K N D A

spDnm1/1-781 130 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q VR E E V N IN N KIY L I L IPV D R LIM YISK A N L A A KL TR T A T S E ddDynA/1-853 98 EI T R G KG PI L S V NLTL DLPG TK G QP DIE Q EIR D D MT N IS N KIY P V V I VPV D IR MVM YI E IR K AQ H T Q R A K

rnDynI/1-864 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL SP S LAN DA KIAKE IG I D DV KLLPLR YIGVVKE C Q Q E R EN R btDyn1/1-856 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL SP S LAN DA KVAKE IG I D DV KLLPLR YIGVVKE C Q Q E R EN R hsDyn1/1-864 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL SP S LAN DA KVAKE IG I D DV KLLPLR YIGVVKE C Q Q E R EN R mmDyn1/1-867 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL SP S LAN DA KIAKE IG I D DV KLLPLR YIGVVKE C Q Q E R EN R ggDyn1/1-865 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL SP S LAN DA KVAKE IG I D DV KLLPLR YIGVVKE C Q Q E R EN R drDyn1/1-843 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LLL SP S LAN DA KIAKE IG I D DI KLLPLR YIGVVKE C Q L E R EN R rnDyn2/1-870 166 AV AN D S L VDP G RT V TKLDLMD GT A L G S LIL TP LAN DA KLAKE IG I D DV KLLPLR YIGVVRE S M Q L E R EN R rnDyn3/1-869 166 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL TP T LAN DA KLAKE IG I D DV KLLPLR YVGVVRE C Q L E R EN R dmDyn/1-877 161 AV AN D S L VDP G RT V TKLDLMD GT A L G T LIL TP T LAN DA KLAKE IG I D DI KLLPLR YIGVVKE C Q V E R EN R ceDyn1/1-830 168 AV AN D S L VDP G RT V TKLDLMD GT A L G T LIL TP S LAT DA KLAKE IG L D EI KL LR YVGVVRE C Q L E R EN FT R

rnDrp1/1-755 189 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIL T T MAT EA KI RE L I D DV RVIPVK IGVVNP S A S D R A A M MG L I btDrp1/1-749 189 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIL T T MAT EA KI RE L I D DV RVIPVK IGVVNP S A S D R A A M MG L I hsDrp1/1-736 176 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIL T T MAT EA KI RE L I D DV RVIPVK IGVVNP S A S D R A A M MG L I mmDrp1/1-742 182 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIL T T MAT EA KI RE L I D DV RVIPVK IGVVNP S A S D R A A M MG L I drDrp1/1-691 175 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIL T T MAT EA KVARE L V D DV RVIPVK IGVVNP S A D R A A M MG L L scDnm1/1-757 205 AV AN D S L VDP G RT V TKLDLMD GT A L G N LIL SP LVN E KLARE IG I DI KM PLK FVGVVTP C V S Q K S N L SG Y L

spDnm1/1-781 200 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIL SP IVN E KLAR IG L DI RV PLK FV VRP S F G S K K Q N M SG Y L AT ddDynA/1-853 168 AV AN D S L VDP G RT V TKLDLMD GT A L G N IIV TP T LAN DA LAKE IG I D EV RVIPL FIGVIKQ A Q E K K M TG TL

shibire ts2

K142A

R66AK44AS45N

T65A/D/H/F

T141Q/A/D

S41A/D

sushi R59C

L12N F20N Q40E

D180A

SUPPLEMENTARY INFORMATION

2 | W W W. N A T U R E . C O M / N A T U R E

RESEARCH

αC αC’’ CGTPase ...

... CGTPase α1N α1C1

α1C2 α2 ...

α3

G397D (scDnm1 G436D)

Dynamin-specific loop

shibire ts1 rnDynI/1-864 236 NR Q DI K E Y GT L L L HIR LP S K I L FF HP R LA R Y K N D LRNKLQ LL K DG D TAA AA RK LS S H D M P Q V QQ TN T G SQ

btDyn1/1-856 236 NR Q DI K E Y GT L L L HIR LP S K I L FF HP R LA R Y K N D LRNKLQ LL K DG D TAA AA RK LS S H D M P Q V QQ TN T G SQ hsDyn1/1-864 236 NR Q DI K E Y GT L L L HIR LP S K I L FF HP R LA R Y K N D LRNKLQ LL K DG D TAA AA RK LS S H D M P Q V QQ TN T G SQ mmDyn1/1-867 236 NR Q DI K E Y GT L L L HIR LP S K I L FF HP R LA R Y K N D LRNKLQ LL K DG D TAA AA RK LS S H D M P Q V QQ TN T G SQ ggDyn1/1-865 236 NR Q DI K E Y GT L L L HIR LP S K I L FF HP R MA R Y K N D LRNKLQ LL K DG D QAA AA RK LS A H D M P Q V QQ TN T G SQ drDyn1/1-843 236 NR Q DI K E Y GT L L L HIR LP S K I M FF HP R LA R Y K N D LR KLQ LL K DG D NAA AA RK LS S H D M P Q T QQ TN T R A SQ rnDyn2/1-870 236 NR Q DI K E Y GT L L L HIR LP S R I L FF HP R MA R K N E LRSKLQ LL K EG D RAA AA RK LS A H D M PH Q T QQ TN S T SQ rnDyn3/1-869 236 NR Q DI K E Y GT L L L HIR LP S K I M FF HP R IA R K N D RNKLQ LL K DG D KAA LA RK LS A H D M PH Q V QQ TN T NF GQ dmDyn/1-877 231 NR Q DI K E Y GT L L L HIR LP S R I L FF HP R MA R Y R N D LR KLQ ML K EG D HQA AA RK LS S H D L P Q V QQ TN T G D KQ ceDyn1/1-830 238 NR Q DI K E Y GT L L L HIR LP R I L FF HP R MA R Y H N D LR LQ M G K VG D RAA DA RK IS S H D L S Q T QQ TN T T DS KK F

rnDrp1/1-755 259 NR Q DI K E Y GT L L L HIR LP S K V I F K LA R Y R N D LKTRIN AA L NN S TDS RD YA LQK .. PS N N K A T RL MH C E VL btDrp1/1-749 259 NR Q DI K E Y GT L L L HIR LP S K V I F K LA R Y R N D LKTRIN AA L NN S TDS RD YA LQK .. PS N N K A T RL MH C E VL hsDrp1/1-736 246 NR Q DI K E Y GT L L L HIR LP S K V I F K LA R Y R N D LKTRIN AA L NN S TDS RD YA LQK .. PS N N K A T RL MH C E VL mmDrp1/1-742 252 NR Q DI K E Y GT L L L HIR LP S K V I F K LA R Y R N D LKTRIN AA L NN S TDS RD YA LQK .. PS N N K A T RL MH C E VL drDrp1/1-691 245 NR Q DI K E Y GT L L L HIR LP S K V I F K LA R Y R N D LKTRIN A L NN S ADS RD HG LQK .. PS N N K A T RL MH C E VLS scDnm1/1-757 275 NR Q DI K E Y GT L L L HIR LP S V L YF HP R I K Y K N D IKTKLN I Q QLN T EES DK ED RK V T ST C R A L QT LS K D TL S

spDnm1/1-781 270 NR Q DI K E Y GT L L L HIR LP S H M L FF HP R I R Y K S E IK RLS I S VS S RDA QS RS EH A T KD C P A T NL VS R D A TL SddDynA/1-853 238 NR Q DI K E Y GT L L L HIR LP S K I L YF HP K IA R Y K N D LK KVS L E IA S RES KS IL KN I S N S A S T KL MF T D V KM S

rnDynI/1-864 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S IDT RI IFHE FP L IEKE DE KNFRP DPAR K Q AV EKR GDQ. Y S NR R FE btDyn1/1-856 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S IDT RI IFHE FP L IEKE EE KNFRP DPAR K Q AV EKR GDQ. Y S NR R FE hsDyn1/1-864 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S IDT RI IFHE FP L IEKE EE KNFRP DPAR K Q AV EKR GDQ. Y S NR R FE mmDyn1/1-867 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S IDT RI IFHE FP L IEKE DE KNFRP DPAR K Q AV EKR GDQ. Y S NR R FE ggDyn1/1-865 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S IDT RI IFHE FP L IEKE EE KNFRP DPAR K Q AV EKR GDQ. Y S NR R FE drDyn1/1-843 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S IDT RI IFHE FP L IEKE EE KNFRP DPSR K Q SV EKR GDQ. Y S NR R FE rnDyn2/1-870 306 LQ I G EL GGA S V Y D KT AL MVQ F DF E S VDT RI IFHE FP L LEKE EE KNFRP DPTR K Q GV EKR GDQ. L S NR R FE rnDyn3/1-869 306 LQ I G EL GGA S V F E KT AL MVQ F DF E S VDT KI IFHE FP I IEHE EA KNFKP DPTR K Q AV EKR GDQ. L S NR R FE dmDyn/1-877 301 LQ I G EL GGA T V F KT AM MIQ DF E S V T KI IFHE I LEKE EE KHFQPGDASI K QLQS ERT GSAL N N S NR RLRFE ceDyn1/1-830 308 LQ I G EL GGA V Y KT AL MVT F D E S V T RI LFHE FP IAMEKD AE KNYQPNDPGR K Q NA IERS SAKL S N S NR R FE

rnDrp1/1-755 327 LQ I G EL GGA Q L Y D KS L LIT F EY E T IET RI IFHE FG L YQSL NS GEPVD .... AT K AT CNT AKY. S C CY T RT btDrp1/1-749 327 LQ I G EL GGA Q L Y D KS L LIT F EY E T IET RI IFHE FG L YQSL NS GEPVD .... AT K AT CNT AKY. S C CY T RT hsDrp1/1-736 314 LQ I G EL GGA Q L Y D KS L LIT F EY E T IET RI IFHE FG L YQSL NS GEPVD .... AT K AT CNT AKY. S C CY T RT mmDrp1/1-742 320 LQ I G EL GGA Q L Y D KS L LIT F EY E T IET RI IFHE FG L YQSL NS GEPVD .... AT K AT CNT AKY. S C CY T RT drDrp1/1-691 313 LQ I G EL GGA Q L Y D S L LIT F EY E T IET RI IFHE FG L YQSL SS GEPVE ....M AT K AT CNT AKY. A C CY T RT scDnm1/1-757 345 LQ I G EL GGA Q L Y R LV LMN F F D T I T RI IY FG L TEQE AR GGVGATTNES AS K STN ISS SSD. N K C YY NNV NS

spDnm1/1-781 340 LQ I G EL GGA Q L Y R IL AMN F F D N I T RL IF F L TQQQ NN GDFKLSDQSQ GI R ANT IAS SSN. P K S YS NNV TTA ddDynA/1-853 308 LQ I G EL GGA L Y D LL IIT F F D L N RI IF E Y VDVQGE ST GDPLY TKNSQGA I SSN KDA KLTD. S N Y SY N I SHC

rnDynI/1-864 375 N G R LF F K Q P V E ME EI AIK I PDL EA V K V KL E IKCVDM L I CS VK FDEKELRR SY H I TG T A T Q K S VS TST RK .btDyn1/1-856 375 N G R LF F K Q P V E ME EI AIK I PDL EA V K V KL E IKCVDM L I CS VK FDEKELRR SY H I TG T A T Q K S VS TAT RK .hsDyn1/1-864 375 N G R LF F K Q P V E ME EI AIK I PDM E IV K V KI E LKCVDM L V CT VK FDEKELRR SY H I TG T A T K R C IS IST RQ .mmDyn1/1-867 375 N G R LF F K Q P V E ME EI AIK I PDM E IV K V KI E LKCVDM L V CT VK FDEKELRR SY H I TG T A T K R C IS IST RQ .ggDyn1/1-865 375 N G R LF F K Q P V E ME EI AIK I PDM E IV K V KI E LKCVDM L V CT VK FDEKELRR SY H I TG T A T K K C IS INT RQ .drDyn1/1-843 375 N G R LF F K Q P V E ME EI AIK I PDL EAIV K I KL LKCIDM L I CS VK FDEKELRK SY H I TG T A Q NG C VS TST RK .rnDyn2/1-870 375 N G R LF F K Q P V E ME EI AIK I PDL EAIV K V KL E LKCVDL L V CT VK FDEKDLRR SY H V TG T A V K C IQ IST RQ .rnDyn3/1-869 375 N G R LF F K Q P V E ME EI AIK I PDM EAIV K I KL LK VDL L V CT VK FNEKELRR SY H I TG T A V KG S S MQ INT KK .dmDyn/1-877 371 N G R LF F K Q P V E M EI AIR I PDM EAIV R I L E IKCVDL L V CT VK ACDEKELRR SF H I VG T A AL K V VQ SVV RM .ceDyn1/1-830 378 N G R LF F K Q P V E ME EI AIR I PDM EAIA K I RL E LKCVDL L I C VK IDEKEMRK QY H I VG T A T K S VN ANV RQ A.

rnDrp1/1-755 392 N G R LF F K Q P V E VD DI AIR A PEV ELLV R I RL E LRCVEL M I CS ES PLGGLNTI LT T P PA V S K E S HE QRI QH NbtDrp1/1-749 392 N G R LF F K Q P V E VD DI AIR A PEV ELLV R I RL E LRCVEL M I CS ES PLGGLNTI LT T P PA V S K E S HE QRI QH NhsDrp1/1-736 379 N G R LF F K Q P V E VD DI AIR A PEV ELLV R I RL E LRCVEL M I CS ES PLGGLNTI LT T P PA V S K E S HE QRI QH NmmDrp1/1-742 385 N G R LF F K Q P V E VD DI AIR A PEV ELLV R I RL E LRCVEL M I CS ES PLGGLNTI LT T P PA V S K E S HE QRI QH NdrDrp1/1-691 378 N G R LF F K Q P V E VD DV AIR A PEV ELLV R V RL E LRCVEL M I CS ES PLGGLTTI LT T P PA V S K E S HE QRI QH NscDnm1/1-757 414 N G R LF F K Q P V E ID DV AIR PEL DLLV I L E RCVEL L C KS PTSNLSVL RT ST P PT V A P KL L SQ YE MKICHK GS

spDnm1/1-781 409 N G R LF F K Q P V E ID DI AI EM DILV L L CVEL L NS PLQNLSTV RT L ST S AT LS A P NL AA CHQ YE MKICHYSGDddDynA/1-853 377 N G R LF F K Q P V E ID DI MR A PEI ELLV K V RL E A CVE L V NN PLEGISLN RTT T P AA I S V E S Q Y YD QRI SQLEA

rnDynI/1-864 444 L NT H F L YPR RE M IVT IR R T E V LI IELAYM N D I A ...EK QQ E ER TH E EGR K Q ML D E GFAN Q..........btDyn1/1-856 444 L NT H F L YPR RE M IVT IR R T E V LI IELAYM N D I A ...EK QQ E ER TH E EGR K Q ML D E GFAN Q..........hsDyn1/1-864 444 L NT H F L YPR RE M IVT IR R T E V LI IELAYM N D I A ...KK QQ E ER TH E EGR K Q ML D E GFAN Q..........mmDyn1/1-867 444 L NT H F L YPR RE M IVT IR R T E V LI IELAYM N D I A ...KK QQ E ER TH E EGR K Q ML D E GFAN Q..........ggDyn1/1-865 444 L NT H F L YPH RE M IVT IR R T D V LI IELAYM N D I A ...KK SQ E ER TH E EGR K Q ML D E GFAN Q..........drDyn1/1-843 444 L NT H F L YP RE M IVT IR R T V LI IELAYM N D I A ...EK AQ M E ER QH D ECR KNQ LL D E GFAN Q..........rnDyn2/1-870 444 L NT H F L YPR RE IVT IR R T D I LI IE YI N D I A ...SK SS ETER TY E EGR K Q LL D QS E GFAN Q..........rnDyn3/1-869 444 L NT H F L FPR E IV IR R T D V LI I V YI N D I A ...KR AN C ETER ANH E EGK K Q LL D Q S E GFAN Q..........dmDyn/1-877 440 L NT H F M YPR RE IIT VR R E I LI ELAYM N D I A ...AK SR ETER TH Q EHSCK Q LL DF E GFAN Q..........ceDyn1/1-830 447 L NT H F M YPR RD L IV MR R I IV ELAYM N D I A ...DT AR E ER VSH E EQIAKQQ GL DY E GFSN E..........

rnDrp1/1-755 462 L NT H F L FPK HD I VVT LR R T E V LV IELAYI D A L YSTQE LR A VE CL K LPV N M HN A K P DACG M..........btDrp1/1-749 462 L NT H F L FPK HD I VVT LR R T E V LV IELAYI D A L YSTQE LR A VE CL K LPV N M HN A K P DACG M..........hsDrp1/1-736 449 L NT H F L FPK HD I VVT LR R T E V LV IELAYI D A L YSTQE LR A VE CL K LPV N M HN A K P DACG M..........mmDrp1/1-742 455 L NT H F L FPK HD I VVT LR R T E V LV IELAYI D A L YSTQE LR A VE CL K LPV N M HN A K P DACG M..........drDrp1/1-691 448 L NT H F L FPK HD I VVT LR R T E V LV IELAYI D A L YSTQE LR A VE SL K LPV N M HN A K P DACG M..........scDnm1/1-757 484 L NT H F L YPK K L VIS LR R T V LI I AYI N L A ...AE AR SM IE EL E LQP RSY ES D HR PN SATE M..........

spDnm1/1-781 479 L NT H F I FPK L VS LR T V LI I AYI N D L A ...SD SH QTA VET DL ENLTP YSF ES A QS P GVQG M..........ddDynA/1-853 447 L NT H F L F K V VVN L H T I LI IE AFI S D V I ...KE SR IN AR IE NL QK KVP KTM EH K T P GGEG FESLYKKQQLQ

L2

L1

G346 (scDnm1 G385D)

R361S

R399A... α2


SUPPLEMENTARY INFORMATION RESEARCH

β1 β2...

...β2 β3 β4 β5 β6 β7...

...β7 αC

α4...

VL1

VL2 VL3

rnDynI/1-864 501 Q S NKK VIR TIN K S ............ R NQM KT..SGNQDEIL...... KGWL NIGIM GG KEYWF.......btDyn1/1-856 501 Q S NKK VIR TIN K S ............ R NQM KA..SGNQDEIL...... KGWL NIGIM GG KEYWF.......hsDyn1/1-864 501 Q S NKK VIR TIN K S ............ R NQM KT..SGNQDEIL...... KGWL NIGIM GG KEYWF.......mmDyn1/1-867 501 Q S NKK VIR TIN K S ............ R NQM KT..SGNQDEIL...... KGWL NIGIM GG KEYWF.......ggDyn1/1-865 501 Q S SKK VIR TIN K S ............ R SQM KA..AGNQDEIL...... KGWL NIGIM GG KEYWF.......drDyn1/1-843 501 Q S KK VIR TIN K ............ R SQIK AA...GNQDEIM...... KGWL NIGIM GGAKEYWF.......rnDyn2/1-870 501 Q S NKK VIR TIN K S ............ R TQL RA..IPNQGEIL...... RGWL NISLM GG KEYWF.......rnDyn3/1-869 501 Q S KK VIR TVS K S ............ R SQVH ST..IGNQGTNLPPSRQI KGWL NIGIM GG KGYWF.......dmDyn/1-877 497 N S NK VIR IQ K S ............ K ENA TGTRQLGNQ.......... KGHMV NLGIM GG RPYWF.......ceDyn1/1-830 504 Q VIR SLS R S ............AKASQG SAKK.NLGNQ.......... KGWL NVSFV G. KDNWF.......

rnDrp1/1-755 522 N N QRR LAR VS K ............ N IEE NR................ ELPSA RD... SSKVPSAL.......btDrp1/1-749 522 N N QRR LAR VS K ............ N IEE NR................ ELPSA RD... SSKVPSAL.......hsDrp1/1-736 509 N N QRR LAR VS K ............ N IEE NR................ ELPSA RD... SSKVPSAL.......mmDrp1/1-742 515 N N QRR LAR S K ............ N IEE NR................ ELPSAG RD... SSKVPSAL.......drDrp1/1-691 508 N N QRR MR SV Q ............ N IEE NR................. ELPT PRDKMAGGA AE..........scDnm1/1-757 541 RK L S Q N ............DDIMKTR RNQ...............E LKSKL Q ENGQTNGI GTSSISS.....

spDnm1/1-781 536 I TV S ............AVVLSRKEQNRLMLSQENDE.......P SSALD KPDGIELYS DPDTSVK.....ddDynA/1-853 514 Q Q Q V N Q T QQNHLQQLQDQY Q QQQ QQQQQQNGINNNQKGDNGNMN NQQNM Q NMNQQNQS NPFLQQQQQGQN

rnDynI/1-864 544 V S E K K G M N Q YK ..... LTAENL WYKDD EKE KYMLSVDNL LRDVEK F ..SSKHIFALF TE RNV DYRQL..EbtDyn1/1-856 544 V S E K K G M N Q YK ..... LTAENL WYKDD EKE KYMLSVDNL LRDVEK F ..SSKHIFALF TE RNV DYRQL..EhsDyn1/1-864 544 V S E K K G M N Q YK ..... LTAENL WYKDD EKE KYMLSVDNL LRDVEK F ..SSKHIFALF TE RNV DYRQL..EmmDyn1/1-867 544 V S E K K G M N Q YK ..... LTAENL WYKDD EKE KYMLSVDNL LRDVEK F ..SSKHIFALF TE RNV DYRQL..EggDyn1/1-865 544 V S E K K G M N Q YK ..... LTAENL WYKDD EKE KYMLPVDNL LRDVEK F ..SSKHIFALF TE RNV DYRQL..EdrDyn1/1-843 543 V S E K K G M N Q YK ..... LTAESL WYKDD EKE KYMPQVDNL FRDVEK F ..SSKHIFALF TE RNV DYRQL..ErnDyn2/1-870 544 V S E K K G M N Q YK ..... LTAESL WYKDE EKE KYMLPLDNL IRDVEK F ..SNKHVFAIF TE RNV DLRQI..ErnDyn3/1-869 550 V S E K K G M N Q YK ..... LTAESL WYKDD EKE KYMLPLDNL VRDVEK F ..SSKHVFALF TE RNV DYRSL..EdmDyn/1-877 538 V S D K K G M S YK ..... LTSESI WYKDE EKE KFMLPLDGL LRDIEQ F SMSRRVTFALF PDGRNV DYKQL..EceDyn1/1-830 543 V S E K K G M YK ..... LMSDSL WYKDD EKE KYMLPLDGV LKDIEG F ..SRNHKFALFYPDGKNI DYKQL..E

rnDrp1/1-755 554 A S E K K G I Q T WR ..... PASQEP PAASA ADG LIQDNRRET NVASAG G .GDGGRIGDGG EP TGN GMLKT..SbtDrp1/1-749 554 A S E K K G V Q T WR ..... PASQEP PAASA ADG LIQESRRET NAASGG G .......GDAV EP TGN GMLKT..ShsDrp1/1-736 541 A S E K K G V Q T WR ..... PASQEP PAASA ADG LIQDSRRET NVASGG G .......GDGV EP TGN GMLKT..SmmDrp1/1-742 547 A E K K G I Q T WR ..... PASQEPPPAASA ADG LIQDNRRET NVPSAG G .......GDGG EP TGN GMLKT..SdrDrp1/1-691 539 G WR .....................................QE GT................GT GML....KscDnm1/1-757 579 K I S T F .........................NIDQDSA NSDYDDDG ........DAE KQ KDK LNYFFGKDK

spDnm1/1-781 582 N F .....SITNKAT EITTLKSDDSAKMQPLDVLASKRYNNAFS..........TETAERKT LSYVFG..AddDynA/1-853 584 N G Q Q F KYPGGPPAQQQP QQPNQLNKGPQNMPPNQSKPSSIPQN PNNNNNNNNNNNR DH QGS FSSFFR..A

rnDynI/1-864 605 E E R P LAC TQE VDSWKASFL AGVY ERVGDKEKASETEENGS..............................btDyn1/1-856 605 E E R P LAC TQE VDSWKASFL AGVY ERVGDKEKASETEENGS..............................hsDyn1/1-864 605 E E R P LAC TQE VDSWKASFL AGVY ERVGDKEKASETEENGS..............................mmDyn1/1-867 605 E E R P LAC TQE VDSWKASFL AGVY ERVGDKEKASETEENGS..............................ggDyn1/1-865 605 E E R P LAC TQE VDSWKASFL AGVY ERVGDKDKASEAEENGS..............................drDyn1/1-843 604 E E R P LAC SQE VDGWKASFL AGVY ERVVEKEKNDAGEENGS..............................rnDyn2/1-870 605 D D R P LAC SQE VDSWKASFL AGVY E......KDQAENEDGA..............................rnDyn3/1-869 611 D D R P LAC SQE VDSWKASLL AGVY DKSFTENDENGQAEN................................dmDyn/1-877 601 E D R P LSC TVE VESWKASFL AGVY EKQETQENGDESASEES..............................ceDyn1/1-830 604 E R P LGCTNLD IDAWKASFL AGVY EKQKAQEDESQQEMEDT..............................

rnDrp1/1-755 616 E E K P KAE LLA E........ SKPI IMPASPQKGHAVNLLDVP.............................btDrp1/1-749 610 E E K P KAE LLA E........ SKPI IMPASPQKGHAVNLLDVP.............................hsDrp1/1-736 597 E E K P KAE LLA E........ SKPI IMPASPQKGHAVNLLDVP.............................mmDrp1/1-742 603 E E K P KAE LLA E........ SKPI IMPASPQKGHAVNLLDVP.............................drDrp1/1-691 552 E E K KGD GQG E........ TKLQSSIPASPQKGHAVNLLDVP.............................scDnm1/1-757 616 KGQPVFDASDKKRSIAGDGNIEDFRNLQISDFSLGDIDDL..............................

spDnm1/1-781 635 NNATRKAMSIDKSSSYPLNDSLSGGDTNHKNNHPLKMTDLSNEVE.........................ddDynA/1-853 652 SPDPSLGQYGGANNSNNSNNPTSPINSSSNSGNNYNTFGGQQSSSSSSQQLQQSSQSQYKTSYNNNNNSS

rnDynI/1-864 645 I L SY D PK M M R E R V IV KTV M I H ..........................DSFMHS DPQLE QV T N D MA N R L T LbtDyn1/1-856 645 I L SY D PK M M R E R V IV KTV M I H ..........................DSFMHS DPQLE QV T N D MA N R L T LhsDyn1/1-864 645 I L SY D PK M M R E R V IV KTV M I H ..........................DSFMHS DPQLE QV T N D MA N R L T LmmDyn1/1-867 645 I L SY D PK M M R E R V IV KTV M I H ..........................DSFMHS DPQLE QV T N D MA N R L T LggDyn1/1-865 645 I L SY D PK M M R E R V IV KTI M I H ..........................DSFMHS DPQLE QV T N D MA N R L T LdrDyn1/1-843 644 I L SY D PK M M R E R V IV KTV I I H ..........................DGFMHS DPQLE QV T N D MA N R L T LrnDyn2/1-870 639 I L SY D PK M M R E R V II KSI M I H ..........................QENTFS DPQLE QV T N D VA N R L T LrnDyn3/1-869 649 I L SY D PK M M R E R V II K I I I H ..............................FS DPQLE QV T N D MS N C R L T LdmDyn/1-877 641 I L SY D PK M R E R V IV KT V I ...............................SSDPQLE QV T N D MK T TR M A MLceDyn1/1-830 644 I L SY D PK M I R E R V II KTI V V H ...............................S DPQLE QV T N D MR T K L A L

rnDrp1/1-755 649 I L SY D PK M L R E I IV KNI V V H ..........................VPVARK SAREQ DC V ER K FL R Q S A FbtDrp1/1-749 643 I L SY D PK M L R E I IV KNI V V H ..........................VPVARK SAREQ DC V ER K FL R Q S A FhsDrp1/1-736 630 I L SY D PK M L R E I IV KNI V V H ..........................VPVARK SAREQ DC V ER K FL R Q S A FmmDrp1/1-742 636 I L SY D PK M L R E I IV KNI V V H ..........................VPVARK SAREQ DC V ER K FL R Q S A FdrDrp1/1-691 585 I L SY D PK M L R E I IV KNI V V H ..........................VPVARK SAREQ DC V ER K FL R Q S A FscDnm1/1-757 656 I L SY D PK M L E K I II I V V ..........................ENAEPP TEREELEC L R V FD REM E Q A CL

spDnm1/1-781 680 I L SY D PK M M D K I L K I V I H ..........................TMALED SEREEVEV L E T FN TR I I Q V LddDynA/1-853 722 I L SY D PK M L E R L IV KNV V I H SNNSSYNRYQDDFYGRGDKLNQVPSIIKAPDD TSKEKFET L E I FN K K S S F

CNM mutants

M534CK535M/A

I533C/AG532C

K562E



RESEARCH

...α4 CGEDEnd structure

rnDynI/1-864 689 N L LY E R A I MI KE I SE L LM S E AQR EML M HAL SI DI T V NT F F AN SCGDQNT E A Q D R Y KE L G NT T STPMPP.PbtDyn1/1-856 689 N L LY E R A I MI KE I SE L LM S E AQR EML M HAL SI DI T V NT F F AN SCGDQNT E A Q D R Y KE L G NT T STPMPP.PhsDyn1/1-864 689 N L LY E R A I MI KE I SE L LM S E AQR EML M HAL SI DI T V NT F F AN SCGDQNT E A Q D R Y KE L G NT T STPMPP.PmmDyn1/1-867 689 N L LY E R A I MI KE I SE L LM S E AQR EML M HAL SI DI T V NT F F AN SCGDQNT E A Q D R Y KE L G NT T STPMPP.PggDyn1/1-865 689 N L LY E R A I MI KD I SE L LM S E AQR EML M HAL NI DI S I NT F H AN SCGDQNT E A Q D R Y KE L G NT T STPMPP.PdrDyn1/1-843 688 N L LY E R A I MI KE I E L LM S E AQR EML M HAL NI DI T I NT Y NA AS SCGDQNT E A Q D R Y KE L G ST T STSLPP.PrnDyn2/1-870 683 N L LY E R A I MI K I E L LM S E AQR DML M HAL NI DI S V NT AF HH AY SSADQSS E A Q D R Y KE L G ST T STPVPP.PrnDyn3/1-869 689 N L LY E R A I MI KD I SE L LM S E AQR EML M AL I DI V NV F N AQ SSEDQNT E V Q D R YQ KE LA G NTVT STPAPP.PdmDyn/1-877 680 N L LY E R A I II KD I E L MM S E ATR EML M RA QI DV V NA F NG AH ASGDQAQ E A S E R Y CKD L G SMAT SSPLPP.PceDyn1/1-830 683 N L LY E R A I IV E M E L LM S AQK EML M HA I EV S L QTG F KD AH QCGDTDA E QIE E R Y CKE LR S NM T GDQPPPLP

rnDrp1/1-755 693 N L LY E R A I LV KD L SE V LL S D AQR EAA M KAL QI EI T L HV T Q GQ KSSLLDD T E M K D L QG S A RE H W.......btDrp1/1-749 687 N L LY E R A I LV KD L SE V LL S D AQR EAA M KAL QI EI T L HV T Q GQ KSSLLDD T E M K D L QG S A RE H W.......hsDrp1/1-736 674 N L LY E R A I LV KD L SE V LL S D AQR EAA M KAL QI EI T L HV T Q GQ KSSLLDD T E M K D L QG S A RE H W.......mmDrp1/1-742 680 N L LY E R A I LV KD L SE V LL S D AQR EAA M KAL QI EI T L HV T Q GQ KSSLLDD T E M K D L QG S A RE H W.......drDrp1/1-691 629 N L LY E R A I LV KD L SE V LL S D AQR EAA M KAL QV EI T L HV S Q GQ KPALLDD T E M N D L QK S A RE H W.......scDnm1/1-757 700 N L LY E R A I LV KD V N V LL AQ V V TL I YC S Q R TK KETLFEE V DQTL D ELC KSLG YKK A SN L............

spDnm1/1-781 724 N L LY E R A I LV KD I N V LL S L V I V AS A Q R SK REDFFDT I DENVK E EKCER LS YNQ NK ST F............ddDynA/1-853 792 N L LY E R A I LV KE I NE V LL S SK M IL I EI QS H Q AA KEELFDE E PQIS KSCKA IE RK NE N RDFRN........

rnDynI/1-864 758 VDDSWLQVQSVPAGRRSPTSSPTPQRR.APAV.P......PARPGS.RGPAPGPP....PA.GSALGGAPbtDyn1/1-856 758 VDDSWLQVQSVPTGRRSPTSSPTPQRR.APAV.P......PARPGS.RGPAPGPP....PA.GSALGGAPhsDyn1/1-864 758 VDDSWLQVQSVPAGRRSPTSSPTPQRR.APAV.P......PARPGS.RGPAPGPP....PA.GSALGGAPmmDyn1/1-867 758 VDDSWLQVQSVPAGRRSPTSSPTPQRR.APAV.P......PARPGS.RGPAPGPP....PA.GSALGGAPggDyn1/1-865 758 VDDSWLQVQSVPSGRRSPTSSPTPQRR.APAV.P......PARPGS.RGPAPGPP....PAGGSALGGAPdrDyn1/1-843 757 VDDSWLQVQRTGSGGRSPATSPTPQRR.APPPGP......PARPGS.RGSAPGPP....AA......GGPrnDyn2/1-870 752 VDDTWLQ........NTSSHSPTPQRRPVSSVHP......PGRPPAVRGPTPGPPLIPMPVGATSSFSAPrnDyn3/1-869 758 VDDSWLQ.....HSRRSPPPSPTTQRR.LTLSAP......LPRPASSRGPAPAIP....SPGPHS..GAPdmDyn/1-877 749 VKNDWLP.SGLDNPRLSPPSPGGVRGKPGPPAQSSLGGRNPPLPPSTGRPAPAIP.......NRPGGGAPceDyn1/1-830 753 MSDY...........RPHPSGPSPVPRPAPAP........PG.....GRQAPMPP........RGGPGAP

rnDrp1/1-755 ......................................................................btDrp1/1-749 ......................................................................hsDrp1/1-736 ......................................................................mmDrp1/1-742 ......................................................................drDrp1/1-691 ......................................................................scDnm1/1-757 ......................................................................

spDnm1/1-781 ......................................................................ddDynA/1-853 ......................................................................

rnDynI/1-864 814 PVP.SRPG.....A.SPDPF.G..PPPQVPSRPNRAPPGVPSR....SGQASPSRPESPRP.........btDyn1/1-856 814 PVP.SRPG.....A.SPDPF.G..PPPQVPSRPNRAPPGVPSQ.PIGSGKSIPS................hsDyn1/1-864 814 PVP.SRPG.....A.SPDPF.G..PPPQVPSRPNRAPPGVPSR....SGQASPSRPESPRP.........mmDyn1/1-867 814 PVP.SRPG.....A.SPDPF.G..PPPQVPSRPNRAPPGVPSL.GAWRLNSPQGKHENRAG.........ggDyn1/1-865 815 PVP.SRPG.....A.SPDPF.G..PPPQVPSRPNRAPPGVPSR....PGKASPSRPESPKP.........drDyn1/1-843 809 PVP.SRPG.....A.SPDPFGG..PPPTVPSRPNRAPPSVPSG.P.........................rnDyn2/1-870 808 PIP.SRPGPQNVFA.NNDPF.S..APPQIPSRPARIPPGIPPGVPSRRAPAAPSRPTIIRP.........rnDyn3/1-869 810 PVP.FRPGPLPPFPNSSDSY.G..APPQVPSRPTRAPPSVPSR....RPPPSPTRPTIIRP.........dmDyn/1-877 811 PLPGGRPG.............GSLPPPMLPSRVSGAVGGAIVQ....QSGANRYVPESMRGQVNQAVGQAceDyn1/1-830 791 PPPGMRPP.....PGAPGGG.GGMYPPLIPTRPG..PGGPPPN....MAPPR..................

rnDrp1/1-755 ......................................................................btDrp1/1-749 ......................................................................hsDrp1/1-736 ......................................................................mmDrp1/1-742 ......................................................................drDrp1/1-691 ......................................................................scDnm1/1-757 ......................................................................

spDnm1/1-781 ......................................................................ddDynA/1-853 ......................................................................

rnDynI/1-864 861 .......PFDL... btDyn1/1-856 .............. hsDyn1/1-864 861 .......PFDL... mmDyn1/1-867 864 .......KARL... ggDyn1/1-865 862 .......PFDM... drDyn1/1-843 .............. rnDyn2/1-870 864 .......AEPSLLD rnDyn3/1-869 863 .......LESSLLD dmDyn/1-877 864 AINELSNAFSSRFK ceDyn1/1-830 ..............

rnDrp1/1-755 .............. btDrp1/1-749 .............. hsDrp1/1-736 .............. mmDrp1/1-742 .............. drDrp1/1-691 .............. scDnm1/1-757 ..............

spDnm1/1-781 .............. ddDynA/1-853 ..............

A738NK694A/E R725A

PRD

R730AI690K I697K

sushi A738T

sushiT749I



Figure S1. Sequence alignments of a representative group of dynamin and Drp1

orthologs. Dynamins and Drp1 orthologs are distinguished by grey shading. The Drp1

orthologs are subdivided into two classes indicated by different degrees of grey shading;

mammalian and Danio rerio Drp1 orthologs (light shading) and fungal and Dictyostelium

discoideum Dnm1 orthologs (dark shading). Sequences are shown with identical

residues highlighted in red boxes with white text; residues conserved in greater than 70

% of the sequences are colored in red. The secondary structure assignments of Dyn1

G397D ΔPRD are indicated above the blocks of text with α-helices represented with

cylinders and β strands represented with arrows. Density missing in the structure is

indicated by dotted lines. Key conserved residues within the GTPase domain are shown

with black arrows above the blocks of sequence along with an annotation of the

sequence motif. The locations of G397D, Dnm1 G385D, the shibire and the

Centronuclear myopathy mutants and of G346 are shown with arrows. Key well-

characterized dynamin mutations are highlighted below sequence blocks, but the

mutations refer to the dynamin 1 sequences.

The sequences used in this alignment are: Rattus norvegicus dynamin 1 (P21575.2),

Bos taurus dynamin 1 (Q08DF4.1), Homo sapiens dynamin 1 (Q05193.2), Mus

musculus dynamin 1 (P39053.2), Gallus gallus dynamin 1 (XP_001233250.1), Danio

rerio dynamin 1 (ABQ82135.1), Rattus norvegicus dynamin 2 (P39052.1), Rattus

norvegicus dynamin 3 (Q08877.2), Drosophila melanogaster dynamin (P27619.2),

Caenorhabditis elegans dynamin 1 (P39055.3), Rattus norvegicus Drp1 (O35303.1), Bos

taurus Drp1 (Q2KIA5.1), Homo sapiens Drp1 (O00429.2), Mus musculus Drp1

(Q8K1M6.2), Danio rerio Drp1 (Q7SXN5.1), Saccharomyces cerevisiae Dnm1

(P54861.1), Schizosaccharomyces pombe Dnm1 (Q09748.1) and Dictyostelium

discoideum Dynamin A (Q94464.2).



RESEARCH

b Atlastin 1 residues 1-4473Q5Epre-fusion formGDP bound

3Q5Dpost-fusion formGDP bound

c Guanylate binding protein1F5NFull lengthGMPPNP bound

2BC9GTPase domainGMPPNP bound

a Bacterial dynamin-like protein1F5NGDP bound

2W6DModel fit to cryo-electron microscopic reconstruction, GDP bound

Figure S2. The crystal structures of BDLP, atlastin 1 residues 1-447 and guanylate binding protein (GBP). Struc-tures are shown with their PDB accession nos and are rendered in the same color scheme as used for Dyn1 G397D ΔPRD. In all cases, the GTPase domains of the left and right structures are shown in the same orientation. a, The structure of the GDP-bound BDLP is a closed hairpin (left) with the membrane-binding “paddle” proximal to the GTPase domain20. On binding to GTP, the structure opens into an extended conformation, based on a fit to the cryo-electron microscopic reconstruction of guanosine-5′-[(β,γ)-imido]triphosphate (GMPPNP)-bound BDLP assembled on a lipid template19. b, Crystal structures of the human atlastin 1 cytosolic domain in the presence of GDP. The two crystal forms are proposed to represent the pre- and post-fusion states of atlastin 1 as indicated. Shown are the crystal structures 3Q5E and 3Q5D24, though similar structures have been reported23. The conserved proline residues between the MD and the GTPase domain are shown. c, The crystal structure of human GBP in complex with GMPPNP and the crystal structure of the GBP GTPase domain dimer in complex with GMPPNP as indicated.



95

130170

72

1:10 1:50

a

b

6 8 1042

10

20

0Sedi

men

tatio

n co

eff.

(S)

Fraction no.

c

BSA 67 67 4.3

LDH 140 35 7.4

Catalase 232 58 11.3

Thyroglobulin 669 334.5 19.4

Standard Protein (kDa) Subunit (kDa) Sed. coeff. (S)

A28

0 (m

AU

)

80 1004020Elution Volume (ml)

100

0

200

60

Dyn1 G397D ΔPRDDyn1 ΔPRD

Figure S3. Characterization of Dyn1 G397D ΔPRD. a, Purification of Dyn1 G397D ΔPRD. Shown is a Coomassie-stained gel of the peak fractions following the final size-exclusion step of the preparation, run as 10-fold (to the left of the molecular weight markers) and 50-fold (to the right) dilutions of the peak. The protein including linker and N-terminal His6 tag has a theoretical mass of 88.2 kDa. b, Comparison of the elution profiles of Dyn1 ΔPRD and Dyn1 G397D ΔPRD from a S200 size-exclusion column under the non-assembly condition used during purification. The green arrow indicates the void volume. c, A typical calibration of a 5-20% sucrose density gradient, in this case shown after centrifugation in 150 mM NaCl at pH 7.0. The standards used to construct the calibration are shown.



RESEARCH

a

b

R361

R361

α4

α1C2

α1C2

α1C1α1C1

α1C1

α1C1

α4

α4

α4

G397D

G397D

R399

R399Close contact

α1N

α1C1 (Dyn1)

α2

α3α4

VL2

L1 loopCGTPase

L2 loop

CGED

NGTPase

GTPase

VL3

PH

L3 loop

α1C (3LJB)

α1C2 (Dyn1)

Dynamin, Drp1 and Dnm1-specific loop

α2

α2

α3

α3

Figure S4. Supplementary analyses of the Dyn1 G397D ΔPRD stalk structure. a, Superposition of the MxA stalk (green, PDB accession no. 3LJB chain B)18 on the stalk of Dyn1 G397D ΔPRD. The structures super-pose with a r.m.s deviation of Cα atoms of 1.7 Å. The coil following α3, present in dynamins, Drp1s and Dnm1, but absent in MxA is shown, as is the split of α1C in Dyn1 G397D ΔPRD. b, L1 and L2 loops from symmetry-related molecules are located in close proximity. Regions lacking density are represented with dotted lines. G346 is shown as a sphere. R361, which disrupts multimerization when mutated to S27, is shown in stick representation. Approximate locations of G397D and R399, which disrupts multimerization when mutated to A27, are shown. Also highlighted is a predicted region of close contact between the appos-ing L2 loops.



a

b

Figure S5. The dynamin PH domain. a, A selection of characterized dynamin 1 PH domain muta-tions. The Centronuclear myopathy (CNM) mutations33, which increase GTPase activity without compromising lipid interaction, are found at the end of the C-terminal α-helix. b, Distribution of the PH domain residues conserved in mammalian Drp1 orthologs.

Q593

M580 E608

R622

E612

E557

R522

I521

P627

N590

K571

K598

Y597

K535

A618T

A618L/W

V625ΔVL3

VL2

VL1

K535 αC

K562

G578

Supplementary Methods Protein expression and purification

Dyn1 ΔPRD and Dyn1 G397D ΔPRD were expressed in Escherichia coli

BL21(DE3)pLysS (Promega) cells as N-terminal His6 fusion proteins using the pET-15b

plasmid. Cells were grown in 2xTY at 37 °C to an OD600 of ~0.6. Protein expression was

induced by addition of 42.5 μM IPTG and cultures were incubated overnight at 21 °C.

The cell pellet was resuspended in 20mM Tris·HCl pH 8.0, 1.93 mM β-mercaptoethanol

and either 500 mM or 350 mM NaCl (resuspension buffer) for Dyn1 ΔPRD or Dyn1

G397D ΔPRD, respectively. Cells were lysed using a microfluidizer (Microfluidics Corp.)

and the lysate was centrifuged at 35,000 rpm for 45 min in a Type 45 Ti rotor (Beckman

Coulter).

Protein was bound to Ni-NTA agarose (Pierce) in batch. After washing with

resuspension buffer supplemented with 30 mM imidazole, beads were packed into a

column and protein was eluted with an imidazole step gradient to a final concentration of

250 mM. Peak fractions were pooled, dialyzed overnight against a reservoir containing

20 mM Tris·HCl pH 8.0, 150 mM NaCl, 1.93 mM β-mercaptoethanol, and applied to a Q-

sepharose (GE Healthcare) anion exchange column. Flow-through fractions were

concentrated and applied to a Superdex 200 16/60 column using the respective

resuspension buffer with Tris·HCl reduced to 5 mM. Dyn1 G397D ΔPRD was

concentrated to ~200 μM prior to freezing. Analysis of Dyn1 G397D ΔPRD by mass

spectrometry indicated that no detectable protein contaminants were present.

Preparation of liposomes and nanotubes

Liposomes contained 10 % PtdIns-4P and 90 % soybean lipid polar extract (Avanti Polar

Lipids). Nanotubes contained 60 % bovine brain galactocerebrosides (Sigma), 30 % 1-

palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) and 10 % PtdIns-

4P. Lipids in chloroform were mixed in a glass tube with methanol added to keep the

solution colorless. The mixtures were dried using a stream of nitrogen gas, and

subsequently under vacuum for at least 30 min. Liposomes and nanotubes were

rehydrated to lipid concentrations of 0.5 or 1 mg/ml by addition of “dynamin buffer” (25

mM HEPES pH 7.0, 25 mM PIPES pH 7.0, 150 mM NaCl). After rehydration, liposomes

and nanotubes were extruded through a 1 μm Nuclepore membrane (Whatman).

SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature10441

WWW.NATURE.COM/NATURE | 10

Biochemical assays for GTPase activity and self-assembly

GTPase assays using the GTP regenerating system were performed essentially as

described16, except reaction mixes without GTP were incubated at 37 °C for 10 min prior

to starting the reactions by addition of GTP and reactions contained a total of 40 mM

NaCl and 117.5 mM KCl. Kinetic parameters were calculated numerically by fitting data

to the Hill Equation using a non-linear least squares method as implemented in

GraphPad Prism.

Self-assembly was measured by light scattering. Samples contained 1 μM protein in

dynamin buffer supplemented with 1 mM MgCl2 and 1.93 mM β-mercaptoethanol. At

indicated time points, GTP or GMPPCP were added to final concentrations of 0.5 and 1

mM, respectively. Changes in light scattering were measured with an Aminco Bowman

Series 2 Luminescence Spectrophotometer with the excitation and emission

monochromators set to 350 nm.

Sucrose density gradient centrifugation

2 ml 5-20 % sucrose gradients in the appropriate buffer were overlayed with 10 μg Dyn1

ΔPRD or Dyn1 G397D ΔPRD in 150 μl buffer supplemented with 5% sucrose and were

centrifuged at 55,000 rpm for 4 hrs in a TLS-55 rotor. Gradients were fractionated by

hand into 10 aliquots and analyzed by SDS-PAGE and silver staining. Band intensities

were quantified with ImageJ44. Sedimentation coefficients were calculated by

interpolation of a straight-line fit of a range of proteins of known mass and sedimentation

coefficient run for each gradient (HMW Calibration Kit – GE Healthcare).

Negative stain electron microscopy

1 μM protein (Dyn1 ΔPRD or Dyn1 G397D ΔPRD) was incubated for 20 min at room

temperature in the presence of 0.25 mg/ml nanotubes, 0.5 mM GMPPCP and 1 mM

MgCl2 in dynamin buffer supplemented with 1.9 mM β-mercaptoethanol in a total volume

of 20 μl. 10 μl were placed on a glow-discharged carbon-coated grid for 1 min and the

grid was stained with a 2 % uranyl acetate solution, blotted and air-dried. Samples were

imaged at 20,000 x with a Phillips CM120 Biotwin Lens transmission electron

microscope (F.E.I. Co.), located in the Electron Microscopy Laboratory, Department of

Pathology and Laboratory Medicine, School of Medicine, UC Davis, operating at 80 kV.



Images were captured using a MegaScan CCD and the Gatan package of Digital

Micrograph software.

Crystallization and structure determination

Initial crystallization conditions were obtained using the High Throughput Screening Lab

service at the Hauptman-Woodward Medical Research Institute (Buffalo, NY)45. Several

conditions produced crystals and the most promising were extensively optimized. As the

initial hits could not be reproduced by vapor diffusion methods, the crystals used in this

work were grown by microbatch at room temperature under light mineral oil (specific

gravity 0.845-0.905) from 3.2 μl droplets containing 52.5 mM Tris·HCl pH 7.7, 175 mM

NaCl, 32.5 mM NaNO3, 20% v/v PEG-400, 0.97 mM β-mercaptoethanol and 31.9 μM

G397D protein. Crystals grew to final dimensions of ~200 x 20 x 20 μm, approximately 1

month after set-up. Crystals were frozen in Paretone-N (Hampton Research) as more

reproducible and higher quality diffraction was obtained than by freezing in the cryo-

protecting mother liquor.

Reflection data were collected at Beamline 8.3.1 at the Advanced Light Source46

(Berkeley, California 94720, USA), using a wavelength of 0.9488 Å. Crystals are

orthorhombic and belong to space group C2221, with axes a = 178.45, b = 191.61 and c

= 60.52 Å. A redundant and complete native dataset was collected to a minimum Bragg

spacing of 3.1 Å (Supplementary Table 3). Reflections were indexed using Mosflm47 and

integrated and scaled using XDS48. Intensity statistics indicated moderate anisotropic

diffraction and the data were ellipsoidally truncated by applying resolution cutoffs along

the principle crystallographic axes of 3.10 Å for b* and c*, and 3.30 Å for a*49.

The structure contains one dynamin molecule per asymmetric unit and was solved by

molecular replacement, using Phaser50, as implemented in Phenix51. Useful template

models were identified by a two-stage search of all protein domain entries in the

Structural Classification of Proteins database, as implemented in Wide Search Molecular

Replacement52. The nucleotide-free rat Dynamin 1 GTPase domain (PDB accession no.

2AKA chain B)30 and the human dynamin PH domains (PDB accession nos 1DYN,

2DYN)11,13 were identified as the strongest model candidates. A subsequent search in

the remaining available space in the asymmetric unit with either chain of the human MxA

stalk (PDB accession no. 3LJB)18 gave a weak rotation and stronger translation solution



that could be improved by trimming to those parts that most clearly fit the density. The

remainder of the molecule became apparent in difference density maps. We completed

the model through iterative cycles of refinement and manual model building in O53 and

COOT54. The structure was refined in Phenix to R/Rfree values of 0.21/0.27 using

individual restrained coordinate and B factor refinement in combination with refinement

of the parameters for 20 TLS groups that were defined using the TLS Motion

Determination server55. “Riding” hydrogen atoms were included during the refinement.

The final model deposited with the Protein Data Bank (PDB accession no. 3ZVR)

consists of residues 5-346, 356-396, 403-496, 518-534, 538-575, 583-628 and 653-743

and was of good quality, as assessed using the MolProbity structure validation

software56 (Supplementary Table 3).

Fitting of the dynamin crystal structure into the cryo-EM reconstruction

Atomic structures were fit into the helical reconstruction of dynamin in the GMPPCP-

bound state determined at approximately 20 Å resolution25. We first derived the helical

conformation of the stalk domains (residues 324-496 and 653-708) by applying a shift

and rotation between two interface 2-dimers observed in linear arrangement of our

crystal lattice to match the geometry of the helix followed by real space refinement in O

(Fig. 4a). Analysis of the density necessitates a conformational change of the GTPase

head domain and the BSE with respect to the stalk when the full-length dynamin crystal

structure is placed on the stalk domains. This is likely because of the different

nucleotide- and assembly-states of dynamin of the cryo-EM helix and crystal structure.

We obtained a good fit of the dimeric GDP•ALF4--bound core GTPase domains (PDB

accession no. 2X2E, residues 32-289)10, when placed into the highest density in the

outer layer of the reconstruction. Guided by additional electron density leading from the

GTPase head domain to the stalk interface 1, we fitted the BSE (residues 5-24, 292-318

and 718-743) as rigid body in an opened-up conformation with respect to the GTPase

domain. Connections between the opened-up BSE and the GTPase domain and the

stalk (residues 24-32, 284-296, 319-323 and 707-718) were reestablished using the

program Modeller57. The BSE conformational change can be described as a flipping-out

motion of the BSE at hinge regions P294 (α-helical kink) and P32 (flexible loop). The PH

domain (residues 518-628) is attached to the dynamin stalk via two flexible linkers and

was therefore fitted as an independent rigid body.



Figures were generated with Pymol58. Dimer interfaces were analysed with LIGPLOT59

and AREAIMOL from the CCP4 suite of crystallographic software60. Alignments were

made using MUSCLE61 and the alignments shown were made using the output of

ESPRIPT62 as a starting point. Secondary structure assignments were made using

DSSP63 through the ESPRIPT web server.



Table S1 Sedimentation coefficients and mass calculation for Dyn1 ΔPRD and Dyn1 G397D ΔPRD

Hydrodynamics (500 mM NaCl, pH 8.0)

Stokes radius (Å) 71.3 61.4

Mass (kDa) 212 180

Subunits 2.4 2.1

Sedimentation Coefficient (S) Dyn1 ΔPRD Dyn1 G397D ΔPRD

500 mM NaCl, pH 8.0 7.2 7.1

150 mM NaCl, pH 7.0 8.9 6.8

30 mM NaCl, pH 7.0 10.0 6.8



Table S2 GTP hydrolysis kcat measurements determined from three experiments (± SEM) with 1 μM protein and 0.1 mg/ml 10% PtdIns-4P liposomes (where relevant) kcat (min-1), 1 μM protein

Protein alone + Liposomes

Dyn1 ΔPRD 5.3 ± 0.3 102.4 ± 5.0

Dyn1 G397D ΔPRD 6.4 ± 1.0 8.7 ± 1.1



Table S3 Data collection, refinement and model statistics

Data collection Space group C2221 Cell dimensions

a, b, c (Å) 178.45, 191.61, 60.52 Resolutiona,b:

b*, c* principal axis (Å) 130.6 – 3.10 (3.18 – 3.10) a* principal axis (Å) 130.6 – 3.30

Unique reflections 18735 (948) Rmerge (%) 6.8 (88.1) I / σI 15.4 (1.9) Completeness (%) 96.7 (66.2) Redundancy 4.1 (4.3) Refinement Asymmetric unit composition:

Dyn1 G397D ΔPRD 1 PEG-400 molecules 2.5 Waters 5 Protein residues 669 Nonhydrogen atoms 5460

Resolution (Å) 95.8 – 3.10 No. reflections 18731 Completeness (%) 96.7 Number of test reflections 956 Test reflections (%) 5.1 Rcryst / Rfree (%) 21.3 / 27.2 Wilson B factor (Å2) 101.7 Model geometry R.m.s deviations:

Bonds (Å) 0.008 Angles (º) 1.087 Dihedrals (º) 16.0

Ramachandran analysis: Favored (%) 90.1 Allowed (%) 8.7 Outliers (%) 1.2

MolProbity scorec 2.20, 99th percentile

(N = 2114, 3.10 Å ± 0.25 Å) a Ellipsoidal resolution cutoffs along the crystallographic reciprocal principle axes were applied49. b Values for highest resolution shell are given in parentheses. c 100th percentile is the best among structures of comparable resolution; 0th percentile is the worst56.



Supplementary References

44 Rasband, W. S. ImageJ. http://imagej.nih.gov/ij/ (2011). 45 Luft, J. R. et al. A deliberate approach to screening for initial crystallization

conditions of biological macromolecules. J. Struct. Biol. 142, 170‐179 (2003). 46 MacDowell, A. A. et al. Suite of three protein crystallography beamlines with

single superconducting bend magnet as the source. J. Synchrotron Radiat. 11, 447‐455 (2004).

47 Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data Joint CCP4 + ESFEAMCB Newsletter on Protein Crystallography, No. 26. (1992).

48 Kabsch, W. Xds. Acta Crystallogr. D 66, 125‐132 (2010). 49 Strong, M. et al. Toward the structural genomics of complexes: crystal

structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 8060‐8065 (2006).

50 McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658‐674 (2007).

51 Adams, P. D. et al. PHENIX: a comprehensive Python‐based system for macromolecular structure solution. Acta Crystallogr. D 66, 213‐221 (2010).

52 Stokes‐Rees, I. & Sliz, P. Protein structure determination by exhaustive search of Protein Data Bank derived databases. Proc. Natl Acad. Sci. USA 107, 21476‐21481 (2010).

53 Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110‐119 (1991).

54 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486‐501 (2010).

55 Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439‐450 (2006).

56 Davis, I. W. et al. MolProbity: all‐atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375‐383 (2007).

57 Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779‐815 (1993).

58 Schrödinger, L. The PyMOL Molecular Graphics System, Version 1.4. www.pymol.org (2004).

59 Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein‐ligand interactions. Protein Eng. 8, 127‐134 (1995).

60 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760‐763 (1994).

61 Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792‐1797 (2004).



62 Gouet, P., Robert, X. & Courcelle, E. ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 31, 3320‐3323 (2003).

63 Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen‐bonded and geometrical features. Biopolymers 22, 2577‐2637 (1983).



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